RP-HPLC-Based Bioanalytical Approach for Simultaneous Quantitation of Cinnarizine and Domperidone in Rat Plasma

Abstract

An accurate, precise and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) bioanalytical approach was developed for the simultaneous estimation of cinnarizine (CIN) and domperidone (DOM) in rat plasma using irbesartan (IRB) as an internal standard (IS). The proposed RP-HPLC approach was validated as per the latest ICH M10 guidelines. The analytes (CIN and DOM) and IS were extracted from plasma samples using the protein precipitation strategy. Chromatographic separation is accomplished by a C₁₈ Sunfire^TM (5 µm, 250 mm × 4.6 mm) analytical column, using an isocratic mobile phase consisting of acetonitrile-methanol in 30:70 proportions at a flow rate of 1 mL/min. The detection of all three constituents was recorded at a wavelength of 270 nm with a UV detector. DOM, CIN and IS were eluted at 3.2, 4.5 and 6.1 min, respectively, utilizing a total run time of 10 min. The lower limit of quantification (LLOQ) was 5 ng/mL for CIN and DOM in rat plasma. The proposed RP-HPLC approach was linear in the 5–200 ng/mL range for CIN and DOM. The recovery of the method was greater than 95%, and the relative uncertainty was less than 2%, indicating that the proposed bioanalytical approach was accurate and precise. The limit of detection was established as 1.1 ng/mL for CIN and 1.7 ng/mL for DOM. The created approach was found to be robust and passed all validation criteria; thus, the proposed RP-HPLC approach can be employed successfully for the simultaneous assessment of CIN and DOM in rat plasma.

1. Introduction

Cinnarizine (CIN) is a piperazine derivative with histamine H1-, dopamine D2- and calcium-channel-blocking activity [1,2]. In addition to treating vertigo/Meniere’s disease, nausea, vomiting and motion sickness, it is also effective for vestibular symptoms due to other origins [2,3]. CIN inhibits vascular smooth muscle cell contractions by inhibiting L-type and T-type voltage-gated calcium channels [3]. CIN may alleviate motion sickness vomiting by binding to dopamine D₂ receptors, histamine H₁ receptors and muscarinic acetylcholine receptors [4]. It works by interfering with signal transmission between the inner ear’s vestibular apparatus and the hypothalamus’ vomiting center [4,5].

Domperidone (DOM) is a specific blocker of dopamine receptors with antiemetic and gastric prokinetic properties, which can be used to relieve motility disorders [6,7]. It is frequently employed as an antiemetic medicine for the temporary relief of nausea and vomiting brought on by Parkinson and anticancer medication. The dopamine-receptor-blocking activity of DOM at the stomach level as well as in the chemoreceptor trigger zone is connected to its antiemetic characteristics. It possesses strong affinity for the chemoreceptor trigger zone’s D2 and D3 dopamine receptors. The peripheral dopamine-receptor-blocking activities of DOM are associated with its gastroprokinetic characteristics [8]. By promoting esophageal and gastric peristalsis and reducing esophageal sphincter pressure, DOM speeds up gastric emptying and shortens small bowel transit time [9]. Both of these drugs are individually available in the market and have the therapeutic potential to treat the conditions of nausea vomiting/motion sickness. There has been a clinical trial performed by Oosterveld wherein he reported that the combination of CIN and DOM is more effective than individual drugs [10]. The combination has found acceptance since then, and many marketed products, such as Stugil tablets by Eris Lifesciences Ltd., Vertigil tablet by Cipla Ltd. and Stetidom tablets by Dr. Morepen Ltd., are available in the market, which have a dose combination of 20 mg of CIN and 15 mg of DOM to produce a synergistic effect. Both drugs (CIN and DOM) belong to biopharmaceutical classification system (BCS) II drugs, which means they have poor solubility and good permeability [1,6]. Considering the synergistic impact of these drugs, their simultaneous quantification in plasma is highly beneficial.

A literature survey reveals that several methods were reported for the simultaneous estimation of DOM and CIN in bulk and dosage forms by the reverse-phase high-performance liquid chromatography (RP-HPLC) method [2,11,12]. Nevertheless, to the best of our knowledge, no RP-HPLC approach for the simultaneous measurement of CIN and DOM in rat plasma has yet to be published. The measurement of CIN and DOM alone in rat or human plasma, however, has been the subject of several pieces of research using HPLC with ultraviolet (UV) and fluorescence detection [13,14]. DOM in human plasma can be detected using an RP-HPLC method that was established in 2000 [13]. The mobile phase was given at a flow rate of 1.4 mL/min and was composed of methanol, water, triethylamine and acetic acid (60:40:0.02:0.3, v/v). A correlation coefficient (R²) value of 0.9998 revealed that the procedure is linear in the range of 1–20 ng/mL. The limit of quantification (LOQ) for DOM was determined to be 1 ng/mL, whereas the limit of detection (LOD) was found to be 0.2 ng/mL [13]. The HPLC method was also created as a bioanalytical approach in a different investigation to estimate the level of CIN in human plasma. The mobile phase was given at a flow rate of 1 mL/min and included 0.01 M ammonium dihydrogen phosphate buffer of pH 4.2 and a 0.038% triethylamine and acetonitrile 25:75 (v/v) mixture. Between a 1 and 100 ng/mL concentration range, the calibration curve was linear. CIN’s LOD was established to be 1.25 ng/mL. The procedure was validated for accuracy, precision, specificity, recovery and stability. For the pharmacokinetic analysis of CIN in human plasma samples, this approach was found to be accurate, precise, straightforward and reliable [14]. CIN and its metabolites were estimated in human plasma using a liquid chromatography tandem mass spectrometry (MS)/MS (LC-MS/MS) approach in 2018 by Mandal et al. CIN and the internal standard (metoprolol) had chromatographic elution times of 2.32 and 2.05 min, respectively, in a very brief run time of 3.50 min [15]. In 2016, Khan et al. created an easy, quick and accurate RP-HPLC/UV technique employing tenofavir as the internal standard for the simultaneous detection of DOM and itopride in pharmaceutical samples and human plasma. Pumped at a flow rate of 1.5 mL/min, the mobile phase was a mixture of water (pH 3.0) and acetonitrile (65:35 v/v), and peaks were observed at a wavelength of 210 nm. This approach was linear in the 20–600 ng/mL range. The LOD and LOQ for DOM were 5 and 10 ng/mL, respectively. DOM fast-dissolving tablets were successfully analyzed in vivo using this newly established technique in healthy human volunteers [16]. Over the last decade, numerous RP-HPLC methods were developed for the simultaneous estimation of CIN and DOM from their market formulations [2,11,12]. However, these methods are only applicable to the quantification of these drugs in nonbiological fluids. Over time, a few RP-HPLC approaches have also been established for the quantification of CIN and DOM in biological fluids, but only for individual quantification, as these approaches are not able to quantify these two drugs simultaneously. To understand the pharmacokinetic profile of this drug combination, their simultaneous quantification in biological fluid, especially in plasma, is a prerequisite. To accomplish this goal, a simultaneous RP-HPLC-based bioanalytical approach was created and validated for the measurement of these medications in rat plasma. In this current research, successful validation is performed according to the ICH guidelines (M10) 2019 in terms of parameters including the lower limit of quantification (LLOQ), linearity, accuracy, precision, matrix specificity and drugs’ stability in plasma [17,18].

The simultaneous assessment of a mixture of CIN and DOM in rat plasma has not been reported. Therefore, for the purpose of determining CIN and DOM in plasma simultaneously, we created and validated an isocratic HPLC assay with UV detection. The technique may be effective for pharmacokinetic profile determination, active ingredient identification in tablet dosage forms and drug monitoring.

2. Materials and Methods

2.1. Materials and Equipment

CIN and DOM were provided as complimentary samples from Ankur Pharmaceuticals Ltd. (Baddi, India). Irbesartan (IRB) was obtained from Helix Pharmaceutical (Nalagarh, India). HPLC-grade acetonitrile and methanol were provided by E-Merck (Mumbai, India). In this work, triple-distilled water that has been purified using a Milli-Q water purification system to make it HPLC-grade water (conductivity < 1.0 µS/cm and resistivity = 18.2 MΩ) was used. For quantitative analysis, an HPLC system (Shimadzu SPD-20A, Tokyo, Japan) with a UV/visible detector and a 20 µL loop (Rheodyne) outfitted with an LC-20AT pump was employed. The cooling centrifuge and vortex mixer were from REMI (New Delhi, India).

2.2. Animals

The current investigation utilized six Wistar male rats. All of the rats ranged in age from 6 to 8 weeks, and they were all between 250 and 300 g in weight. The rats were housed in husk-lined polypropylene cages with a 12:12 light: dark cycle at a 25 °C temperature with a relative humidity of 55%. Water and a typical pellet meal were given to the animals as food. The Pinnacle Biomedical Research Institute’s Institutional Animal Ethics Committee (PBRI/IAEC/29-03/010), Bhopal, India, gave its approval to the study protocol.

2.3. Chromatographic Conditions

A bioanalytical approach was created for the simultaneous measurement of CIN and DOM in rat plasma following the ICH M10 recommendations [17,18]. The separation of CIN and DOM was achieved using a reverse-phase analytical column (C-18 Sunfire^TM, 5 µm; 250 × 4.6 mm) and was maintained at 30 ± 1 °C. Acetonitrile and methanol were the components of the mobile phase in a ratio of 30:70 v/v, which was pumped at a flow rate of 1 mL/min in an isocratic mode. IRB served as an internal benchmark (IS). A sample volume of 20 μL was injected in triplicate onto the HPLC column. A UV/Vis detector operating at 270 nm was used to find both analytes and the IS.

2.4. Preparation of Blank Plasma

Due to the reduced steps required to extract the drug from the matrix, protein precipitation was the chosen method of separation [19,20]. To separate the plasma proteins, 1 mL of plasma was mixed with 2 mL of a mixture of methanol and acetonitrile (1:1 v/v). The mixture was then agitated for 5 min. The supernatant was taken out and spun in a cooling centrifuge for 15 min at 10,000 rpm. The supernatant was filtered with a syringe filter before being transferred to a volumetric flask with a 100 mL capacity and diluted with the mobile phase to obtain a total of 100 mL of the mobile phase, which was spiked with 1% deproteinized plasma [21].

2.5. Preparation of Standard Stock Solution

CIN and DOM (each 100 mg) were weighed precisely and transferred to individual 100 mL volumetric flasks containing 20 mL of the mobile phase. The solutions were sonicated for 5 min, and the volume was adjusted to 100 mL with the mobile phase to yield a 1000 μg/mL concentration of CIN (solution A) and DOM (solution A₁). To achieve a concentration of 100 μg/mL of each drug, a 10 mL aliquot was withdrawn from each flask into a 100 mL standard volumetric flask, and the volume was adjusted to 100 mL using the mobile phase to obtain solution B and B₁. In addition, a 10 mL aliquot from each solution, B and B_1, was transferred to 100 mL standard volumetric flasks, and the volume was adjusted to 100 mL using the mobile phase to yield a 10 μg/mL concentration of CIN and DOM (solution C and C₁). A total of 10 mL of aliquots from solution C and C₁ were transferred to a 100 mL standard volumetric flask, and the volume was adjusted to 100 mL with the mobile phase to achieve a concentration of 1 μg/mL (1000 ng/mL) (solution D and D₁). By adding several stock solutions to rat plasma, calibrations and quality control solutions were created. For CIN and DOM in plasma, the calibration curve range was 5–200 ng/mL.

2.6. Preparation of Stock Solution of IS

IRB 10 mg was precisely weighed, dissolved in the mobile phase and transferred to a volumetric flask of 100 mL. The solution was sonicated for 15 min and diluted with the mobile phase to create a solution with a concentration of 100 μg/mL (solution E). To obtain a final solution with a concentration of 10 μg/mL (solution F), an aliquot of 10 mL from solution E was withdrawn in a 100 mL standard volumetric flask, and the volume was adjusted to 100 mL using the mobile phase.

2.7. Method Validation Procedures

2.7.1. LLOQ

Five plasma replicates spiked with CIN and DOM at LLOQ were used to validate the LLOQ. According to US FDA guidelines, LLOQ is estimated as 5% of peak plasma concentration (C_max). Published studies showed plasma C_max levels of CIN and DOM up to 100 ng/mL. The coefficient of variation (CV) needs to be under 20%. The lowest calibration standard is known as LLOQ. Furthermore, the LLOQ sample’s analyte signal needs to be at least five times that of a blank sample [22].

2.7.2. Method Specificity

A total of 0.5 mL of blank plasma with a concentration of 1 µg/mL was pipetted out and diluted to 100 mL, obtaining a blank plasma concentration of 5 ng/mL. The method specificity was assessed by injecting 20 µL of this plasma as a blank sample into the HPLC system. Moreover, 5 ng/mL solutions (at the LLOQ level) for CIN and DOM were individually produced from the stock solution in the mobile phase with the spiked matrix. The 50 ng/mL solutions of CIN-DOM (with plasma) were made by diluting 0.5 mL of the 1 µg/mL stock solutions D and D1 to 10 mL. From these solutions, a 1 mL aliquot was pipetted into two 10 mL standard volumetric flasks, 1 mL of the IS from its stock solution (100 µg/mL) was added and the volume of each flask was adjusted to 10 mL to achieve a final concentration of 5 ng/mL for CIN and DOM and 10 µg/mL for the IS. At 270 nm, 20 µL of these solutions were put into the HPLC system and evaluated [23].

2.7.3. Calibration Curves

The seven-point calibration curves for CIN and DOM in the range of 5–200 ng/mL were prepared using aliquots (0.05, 0.1, 0.25, 0.5, 1.0, 1.5 and 2.0 mL) from stock solution (D and D1) having a 1 µg/mL concentration of both drugs. To each of these dilutions, 1 mL of solution F (IRB, 10 µg/mL) and 0.5 mL of rat plasma were added. To obtain concentrations of 5, 10, 25, 50, 100, 150 and 200 ng/mL, the samples were reconstituted to 10 mL using the mobile phase and injected into the HPLC system for measurement at 270 nm. To determine the linearity of the analytical approach, the slope, intercept and R² of the calibration curves (peak area versus concentration) were calculated [24].

2.7.4. Accuracy Study

A study of accuracy was carried out based on the absolute recovery at each of the four distinct quality control (QC) levels for both samples (CIN and DOM). These levels are labeled as the lower limit of quantification (LLOQ), lower quantified concentration (LQC), medium quantified concentration (MQC) and high quantified concentration (HQC). The values for the LLOQ, LQC, MQC and HQC were 5, 15, 100 and 150 ng/mL, respectively (for CIN and DOM each). The IS was added to all of these solutions, and its concentration was maintained at 10 µg/mL throughout. The accuracy was measured in terms of the percentage of CIN and DOM that was successfully recovered from the solution. The experiment was repeated six times, and the average data were recorded. By using equation 1, we were able to determine the percentage of absolute recovery. The mean, standard deviation (SD) and % CV were calculated at each QC level. Accuracy within 85–115% of the nominal values was considered acceptable [18].

$$\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{l}\mathrm{u}\mathrm{t}\mathrm{e} \mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{y}\left(\%\right)=\frac{\mathrm{A}\mathrm{c}\mathrm{t}\mathrm{u}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{e}\mathrm{c}\mathrm{o}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}}{\mathrm{T}\mathrm{h}\mathrm{e}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}}\times 100$$

(1)

2.7.5. Precision Studies

The intraday, interday and interanalyst precisions were determined at four distinct QC levels, i.e., LLOQ, LQC, MQC and HQC. The IS was added to each of these solutions, and its concentration was maintained at 10 µg/mL. In order to conduct an intraday precision (repeatability) study, the solutions of LLOQ, LQC, MQC and HQC were injected into six replicates on the same day. The same solutions were injected on three distinct days to determine interday precision. To determine the interanalyst precision, these samples were administered in six replicates by three different investigators on the same day. They had their mean, SD and % CV computed [25].

2.7.6. LOD and LOQ

Equations (2) and (3) were used, respectively, to obtain the LOD and LOQ for CIN and DOM using the “linear regression equation based on the standard deviation of the intercept and the slope” [26].

$$\mathrm{L}\mathrm{O}\mathrm{D}=\frac{3.3\mathrm{\sigma }}{\mathrm{S}}$$

(2)

$$\mathrm{L}\mathrm{O}\mathrm{Q}=\frac{10\mathrm{\sigma }}{\mathrm{S}}$$

(3)

Here, σ is the standard deviation of the response and S is the slope of the calibration curve.

2.7.7. System Suitability

For the purpose of determining the system appropriateness characteristics, which include the theoretical plates number, tailing factor, resolution and height equivalent to a theoretical plate (HETP), six replicates were injected with the lowest concentration of drugs in the calibration curve (LLOQ, i.e., 5 ng/mL).

2.7.8. Stability Study

Plasma samples spiked with CIN and DOM were subjected to three freeze–thaw cycles, short-term stability studies and long-term stability studies. For freeze–thaw stability, 3 mL of plasma was collected in a single vial. To achieve a concentration of 1000 µg/mL, 3 mg of CIN and DOM were added to this vial, and the mixture was vortexed for 5 min. This test tube was stored at −20 °C in a deep freezer. After the sample was frozen, the test tube was removed and allowed to defrost at room temperature. A total of 1 mL of plasma was extracted from the thawed samples (cycle 1), and the remaining 2 mL was placed in the deep freezer for the second cycle. The drug-containing plasma (1 mL) was precipitated, and the supernatant was centrifuged. After centrifugation, the clear, translucent supernatant was extracted and diluted to 1000 mL with the mobile phase to reach a 1 µg/mL concentration. In addition, dilutions were produced to obtain concentrations of 15 ng/mL (LQC), 100 ng/mL (MQC) and 150 ng/mL (HQC). Likewise, the remaining frozen plasma sample (2 mL) was removed, thawed at room temperature and 1 mL was extracted (cycle 2), while the remaining 1 mL of thawed plasma was returned to the deep freezer. It was taken out and thawed after it had frozen (cycle 3). As in cycle 1, the method was repeated in cycles 2 and 3 to prepare LQC, MQC and HQC samples. The IS was added to all of these solutions at a concentration of 10 µg/mL. All dilutions were produced in triplicate, injected into HPLC and evaluated. For each concentration, the mean, SD and % CV were computed [18,27].

Similarly, the short-term stability of plasma samples spiked with CIN and DOM at room temperature was assessed. The stability was tested 1 h, 2 h and 3 h before extraction. In the short term, 3 mL of plasma was placed in a vial, 3 mg of CIN and DOM were added (to achieve a concentration of 1000 µg/mL) and the mixture was vortexed for 5 min. Room temperature was maintained for the vial. After each interval, a sample (1 mL) was taken, drugs were removed from plasma and processed for the manufacture of LQC, MQC and HQC samples, and the IS (10 µg/mL) was added. All dilutions were produced in triplicate, injected into HPLC and evaluated. For each concentration, the mean, SD and % CV were computed. For long-term stability, 1 mL of plasma was obtained in three vials, and 1 mg of CIN and DOM were added to each vial (to achieve a concentration of 1000 µg/mL). The mixture was vortexed for 5 min, and all three vials were frozen at −20 °C. After one, two and three weeks, the three vials were removed from the freezer. After each interval, the medicines were removed from plasma and processed for the preparation of LQC, MQC and HQC samples, and the IS (10 µg/mL) was added. All dilutions were produced in triplicate, injected into HPLC and evaluated. For each concentration, the mean, SD and % CV were computed [28].

3. Results and Discussion

3.1. Determination of LLOQ

Based on the results, the LLOQ of CIN and DOM was found at 5 ng/mL with %CV values of 3.78% and 3.61%, respectively. These values of %CV for both drugs met the requirements (i.e., not more than 20%); hence, this concentration (5 ng/mL) is elected as the LLOQ. The chromatogram is shown in Figure 1.

3.2. Chromatograms of the Mixture Containing DOM, CIN and IRB

The chromatogram of DOM, CIN and IRB prepared in the mobile phase is shown in Figure 2. Compounds with structurally similar analogues and similar physiochemical properties and functional groups could be used as ISs according to the literature [29,30]. CIN and DOM belong to BCS class II drugs [1,6]. IRB also belongs to BCS class II drugs, having a similar solubility and permeability profile. Furthermore, the molecular weight of IRB is in the same range as that of analytes. Both the solubility and molecular weight of analytes play a vital role in the separation and retention time of analytes [29]. As a result, we have selected IRB as the IS on the bases of physiochemical properties [29,30]. The retention times for DOM, CIN and IRB were found to be 3.20, 4.57 and 6.1 min, respectively.

3.3. Specificity Studies

Figure 3 displays the chromatogram of blank plasma. It was clear that the plasma matrix had no impact on the quantification of any drug extracted from plasma because there was no peak at the retention times (RTs) of DOM, CIN and IRB. Similar results were obtained when blank rat plasma was spiked with CIN and DOM at LLOQ (5 ng/mL) and blank rat plasma was spiked with IRB at 10 µg/mL, demonstrating the lack of any plasma peak in the retention periods of both drugs (Figure 3). Thus, the method is considered specific and selective.

3.4. Calibration Curves for CIN and DOM

Excellent linearity was evident in the calibration curves for both drugs produced in plasma, with R² values for CIN and DOM of 0.999 and 0.998, respectively. The linearity range was 5–200 ng/mL for both drugs. The LLOQ for CIN and DOM was found to be 5 ng/mL at the lowest concentration with a CV value of less than 20%.

3.5. Accuracy Studies

The accuracy was determined by calculating the percentage recovery of both drugs in plasma. The % recovery of CIN and DOM in plasma was measured to be higher than 95%, showing that the proposed bioanalytical approach is accurate (

Table 1. Results of accuracy studies for CIN and DOM.

Levels	Actual Concentration of Drug (ng/mL)	Amount of Drug Recovered in Plasma Sample (ng/mL) N = 5)	Recovery in Plasma (%)	Mean Recovery (%)
CIN
LLOQ	5	Mean = 4.95; SD = 0.04; CV(%) = 0.84	99.00
LQC	15	Mean = 14.88; SD = 0.14; CV(%) = 0.94	99.20
MQC	100	Mean = 99.28; SD = 0.65; CV(%) = 0.65	99.28	99.18
HQC	150	Mean = 148.89; SD = 01.28; CV(%) = 0.86	99.26
DOM
LLOQ	5	Mean = 4.96; SD = 0.04; CV(%) = 0.81	99.20
LQC	15	Mean = 14.86; SD = 0.19; CV(%) = 1.27	99.06
MQC	100	Mean = 99.32; SD = 0.71; CV(%) = 0.71	99.32	99.28
HQC	150	Mean = 149.32; SD = 0.96; CV(%) = 0.64	99.54

). It is worth noting that the % CV in each case was less than 2%, indicating that the findings were repeatable.

3.6. Precision Studies

CIN and DOM in plasma were studied for intraday, interday and interanalyst precision (

Table 2. (a) Results of precision studies for CIN. (b) Results of precision studies for DOM.

(a)
Parameters	Level	Conc.	Analytical Responses (Area)						Mean	SD	CV
		(ng/mL)							(N = 6)
Repeatability	LLOQ	5	11,785	11,781	11,805	11,798	11,878	11,780	11,804.50	37.36	0.32
	LQC	15	12,055	12,043	12,065	12,064	12,033	12,096	12,059.33	21.81	0.18
	MQC	100	13,229	13,156	13,205	13,232	13,205	13,201	13,204.67	27.28	0.21
	HQC	150	13,819	13,862	13,847	13,921	13,793	13,870	13,852.00	44.23	0.32
Intermediate precision (Interday)
DAY 1	LLOQ	5	11,780	11,789	11,786	11,804	11,875	11,787	11,803.50	35.93	0.30
	LQC	15	12,065	12,117	12,041	12,117	12,098	12,038	12,079.33	36.25	0.30
	MQC	100	13,232	13,228	13,205	13,190	13,197	13,168	13,203.33	24.08	0.18
	HQC	150	13,821	13,794	13,809	13,876	13,865	13,810	13,829.17	33.33	0.24
DAY 2	LLOQ	5	11,818	11,797	11,886	11,814	11,769	11,780	11,810.66	41.48	0.35
	LQC	15	12,016	12,034	12,115	12,050	12,178	12,119	12,085.33	62.11	0.51
	MQC	100	13,130	13,198	13,245	13,103	13,342	13,255	13,212.17	87.91	0.67
	HQC	150	13,799	13,621	13,835	13,942	13,851	13,790	13,806.33	105.73	0.77
DAY 3	LLOQ	5	11,758	11,799	11,788	11,770	11,865	11,875	11,809.16	49.30	0.42
	LQC	15	12,087	12,102	12,089	12,158	12,096	12,134	12,111	28.65	0.24
	MQC	100	13,200	13,178	13,297	13,188	13,316	13,197	13,229.33	60.56	0.46
	HQC	150	13,802	13,745	13,915	13,870	13,856	13,789	13,829.50	61.95	0.45
Intermediate precision (Interanalyst)
Analyst 1	LLOQ	5	11,771	11,814	11,790	11,854	11,845	11,789	11,810.50	33.28	0.28
	LQC	15	12,010	12,178	12,056	12,134	12,088	12,005	12,078.50	68.81	0.57
	MQC	100	13,155	13,200	13,118	13,109	13,362	13,158	13,183.67	93.21	0.71
	HQC	150	13,787	13,727	13,739	13,851	13,897	13,875	13,812.67	71.96	0.52
Analyst 2	LLOQ	5	11,845	11,802	11,799	11,765	11,805	11,843	11,809.83	30.16	0.26
	LQC	15	12,047	12,099	12,143	12,005	12,056	12,075	12,070.83	47.20	0.39
	MQC	100	13,189	13,056	13,254	13,168	13,301	13,245	13,202.17	86.02	0.65
	HQC	150	13,717	13,791	13,855	13,870	13,888	13,802	13,820.50	63.44	0.46
Analyst 3	LLOQ	5	11,792	11,882	11,787	11,803	11,805	11,766	11,805.83	39.86	0.34
	LQC	15	11,975	12,186	11,966	12,145	12,005	12,158	12,072.50	100.85	0.84
	MQC	100	13,098	13,139	13,189	13,119	13,254	13,320	13,186.50	86.06	0.65
	HQC	150	13,769	13,936	13,876	13,791	13,881	13,797	13,841.67	65.52	0.47
(b)
Parameters	Level	Con.	Analytical Responses (area), Injections						Mean	SD	CV
		(ng/mL)							(N = 6)
Repeatability	LLOQ	5	5212	5199	5234	5287	5160	5250	5223.67	43.84	0.84
	LQC	15	5778	5828	5789	5765	5686	5770	5769.33	46.62	0.81
	MQC	100	8069	8026	8123	8105	8058	7945	8054.33	63.67	0.79
	HQC	150	9445	9558	9514	9615	9510	9597	9539.83	62.93	0.66
Intermediate precision
DAY 1	LLOQ	5	5160	5230	5209	5188	5274	5190	5208.50	39.67	0.76
	LQC	15	5765	5713	5689	5834	5813	5789	5767.17	56.73	0.98
	MQC	100	8112	8077	8134	8078	8056	8097	8092.33	27.95	0.35
	HQC	150	9456	9555	9634	9542	9578	9617	9563.67	63.46	0.66
DAY 2	LLOQ	5	5213	5189	5243	5217	5278	5158	5216.33	41.65	0.80
	LQC	15	5815	5860	5785	5743	5786	5814	5800.50	39.22	0.68
	MQC	100	8133	8008	8132	8115	8148	8071	8101.17	52.80	0.65
	HQC	150	9603	9456	9743	9612	9575	9548	9589.50	93.81	0.98
DAY 3	LLOQ	5	5250	5189	5148	5245	5215	5198	5207.50	38.06	0.73
	LQC	15	5798	5716	5728	5808	5745	5789	5764.00	39.19	0.68
	MQC	100	8078	8161	8134	8098	8173	8266	8151.67	66.68	0.82
	HQC	150	9645	9466	9589	9607	9469	9614	9565.00	77.66	0.81
Intermediate precision (Interanalyst)
Analyst 1	LLOQ	5	5213	5198	5284	5255	5296	5202	5241.33	42.95	0.82
	LQC	15	5780	5698	5711	5823	5785	5748	5757.50	47.64	0.83
	MQC	100	8104	8156	7978	8132	8204	8164	8123.00	78.48	0.97
	HQC	150	9587	9574	9607	9486	9554	9641	9574.83	52.70	0.55
Analyst 2	LLOQ	5	5232	5277	5178	5207	5227	5166	5214.50	40.27	0.77
	LQC	15	5796	5845	5795	5808	5879	5914	5839.50	49.00	0.84
	MQC	100	8015	8043	8116	7998	8103	8039	8052.33	47.39	0.59
	HQC	150	9474	9624	9565	9516	9597	9675	9575.17	73.03	0.76
Analyst 3	LLOQ	5	5287	5278	5266	5150	5208	5245	5239.00	51.91	0.99
	LQC	15	5769	5830	5790	5824	5716	5864	5798.83	52.31	0.90
	MQC	100	8176	8098	8113	8066	8137	8113	8117.17	37.08	0.46
	HQC	150	9465	9614	9586	9523	9683	9570	9573.50	75.00	0.78

a,b). The % CV of the measured responses of LQC, MQC and HQC samples was less than 2%, suggesting that the procedure was precise.

3.7. LOD and LOQ

In plasma samples, the LOD and LOQ for CIN were determined to be 1.11 and 3.36 ng/mL, respectively. It was determined that the LOD and LOQ for DOM in plasma samples were 1.70 and 5.16 ng/mL, respectively. These results suggested that the approach was sensitive enough to detect both drugs at lower concentrations.

3.8. System Suitability

The tailing factor for both CIN and DOM peaks never exceeded 1 in any of the peaks, which demonstrated good peak regularity (acceptance limits were less than 2). Additionally, the number of theoretical plates for CIN and DOM was more than 50,000 in all runs, whereas the HETP value for CIN was 26.89 and 28.17 for DOM. These parameters’ values ensured excellent column efficiency throughout the development procedure.

3.9. Stability Study of Plasma Samples

For spiking samples of CIN and DOM in plasma, stability studies were performed at three different levels: short term (

Table 3. (a) Short-term stability for plasma samples of CIN and DOM. (b) Freeze–thaw stability for plasma samples of CIN and DOM. (c) Long-term stability for plasma samples of CIN and DOM.

(a)
Level	Mean Area	S.D.	CV	Amount of Drug Recovered in Plasma Sample (ng/mL)	Recovery (%)
CIN
1 h
15	11,924.00	46.13	0.39	14.96	99.74
100	13,141.00	69.16	0.53	100.85	100.85
150	13,831.33	38.85	0.28	149.56	99.71
2 h
15	11,923.67	37.82	0.32	14.94	99.58
100	13,126.67	74.04	0.56	99.84	99.84
150	13,834.00	52.03	0.38	149.75	99.84
3 h
15	11,923.00	46.16	0.39	14.89	99.27
100	13,125.00	71.84	0.55	99.72	99.72
150	13,820.67	37.10	0.27	148.81	99.21
DOM
1 h
15	5514.00	31.22	0.57	15.01	100.08
100	8019.00	63.69	0.79	99.90	99.90
150	9494.00	38.04	0.40	149.88	99.92
2 h
15	5512.33	45.37	0.82	14.96	99.70
100	8016.67	62.00	0.77	99.82	99.82
150	9478.67	57.84	0.61	149.36	99.57
3 h
15	5508.33	38.21	0.69	14.82	98.80
100	8015.00	62.55	0.78	99.76	99.76
150	9458.00	44.51	0.47	148.66	99.11
(b)
Level	Mean Area	S.D.	CV	Amount of Drug Recovered in Plasma Sample (ng/mL)	Recovery (%)
CIN
Cycle 1
15	11,923.67	80.90	0.68	14.94	99.58
100	13,128.00	42.51	0.32	99.93	99.93
150	13,835.33	57.50	0.42	149.85	99.90
Cycle 2
15	11,922.67	76.07	0.64	14.87	99.11
100	13,121.67	49.17	0.37	99.48	99.48
150	13,826.67	71.60	0.52	149.24	99.49
Cycle 3
15	11,922.00	85.11	0.71	14.82	98.80
100	13,126.67	49.54	0.38	99.84	99.84
150	13,803.00	76.73	0.56	147.57	98.38
DOM
Cycle 1
15	5511.67	27.79	0.50	14.93	99.55
100	8015.33	40.50	0.51	99.77	99.77
150	9489.67	59.00	0.62	149.73	99.82
Cycle 2
15	5510.00	31.32	0.57	14.88	99.18
100	8009.67	55.05	0.69	99.58	99.58
150	9473.00	67.98	0.72	149.17	99.45
Cycle 3
15	5507.33	39.53	0.72	14.79	98.57
100	7998.33	44.00	0.55	99.20	99.20
150	9466.33	42.90	0.45	148.94	99.30
(c)
Level	Mean Area	S.D.	CV	Amount of Drug Recovered in Plasma Sample (ng/mL)	Recovery (%)
CIN
Week 1
15	11,924.00	81.61	0.68	14.96	99.74
100	13,125.67	42.50	0.32	99.76	99.76
150	13,834.67	33.65	0.24	149.80	99.87
Week 2
15	11,922.33	87.02	0.73	14.84	98.96
100	13,113.67	59.62	0.45	98.92	98.92
150	13,821.33	70.00	0.51	148.86	99.24
Week 3
15	11,921.67	97.52	0.82	14.80	98.64
100	13,115.33	49.52	0.38	99.04	99.04
150	13,817.33	77.24	0.56	148.58	99.05
DOM
Week 1
15	5511.00	44.93	0.82	14.91	99.40
100	8020.33	46.37	0.58	99.94	99.94
150	9488.33	40.07	0.42	149.69	99.79
Week 2
15	5510.33	44.00	0.80	14.89	99.25
100	8007.67	53.11	0.66	99.51	99.51
150	9474.33	66.16	0.70	149.21	99.48
Week 3
15	5508.67	44.96	0.82	14.83	98.87
100	8008.67	37.50	0.47	99.55	99.55
150	9451.00	54.37	0.58	148.42	98.95

a), freeze–thaw cycles (Table 3b) and long term (Table 3c) at LQC, MQC and HQC levels. The results showed that more than 95 % of both drugs were recovered in all instances, with a % CV of less than 2%. The findings of these investigations pointed to the stress and long-term storage stability of both compounds in rat plasma.

4. Conclusions

CIN and DOM can be estimated simultaneously in rat plasma using a straightforward, isocratic RP-HPLC-based bioanalytical approach that has been devised and effectively validated. The approach that was devised is extremely specialized, accurate, precise and repeatable. The technique uses a very small amount of the plasma sample for analysis and requires no complicated sample preparation. According to the ICH’s most recent M10 recommendations, all validation parameters were within allowable ranges. Most importantly, the new technique has set a standard that researchers can utilize for a variety of in vivo studies, including those on pharmacokinetics, drug distribution, plasma protein binding and drug metabolism.