High-Performance Thin-Layer Chromatographic method for Simultaneous Determination of Some Angiotensin II Receptor Antagonists with Amlodipine in spiked human plasma with UV Detection

doi:10.21203/rs.3.rs-4938215/v1

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High-Performance Thin-Layer Chromatographic method for Simultaneous Determination of Some Angiotensin II Receptor Antagonists with Amlodipine in spiked human plasma with UV Detection

https://doi.org/10.21203/rs.3.rs-4938215/v1

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A simple, sensitive, precise, and cost-effective high-performance thin-layer chromatographic (HPTLC) method has been developed and validated for the simultaneous determination of Amlodipine (AML) and some Angiotensin II Receptor Antagonist (AIIRA) drugs, including Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB), in spiked human plasma. The HPTLC analysis utilized a mobile phase consisting of toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v) on an aluminum-backed layer of silica gel 60 F254. AML demonstrated a linear relationship within the range of 60–600 ng/band, while the AIIRA drugs (OLM, TLM, CAN, LOS, IRB) showed linearity within the range of 90–900 ng/band. The proposed method exhibited good linearity, with correlation coefficients (r) ranging from 0.9939 to 0.9998 for all five studied mixtures. The method was validated according to International Council for Harmonization (ICH) guidelines for linearity, accuracy, precision, robustness, and detection and quantitation limits. AML and the investigated AIIRA drugs were successfully detected and quantified in both bulk drug and plasma samples, yielding high recovery percentages and low standard deviation values.

Amlodipine

AIIRAs

HPTLC

Absorbance

Plasma

Essential hypertension is characterized by an increase in blood pressure without an apparent cause, elevating the risk of cardiac, brain, and renal problems[1]. In developed nations, nearly 90% of the population faces a lifetime risk of developing hypertension (blood pressure greater than 140/90 mmHg). Hypertension is frequently associated with other cardiovascular risk factors, such as aging, obesity, insulin resistance, diabetes, and hyperlipidemia [2]. Drugs from various pharmacological classes with differing efficacy profiles have been developed to treat hypertension. These include calcium channel blockers (CCBs), angiotensin receptor blockers (ARBs), Angiotensin converting enzyme inhibitors, diuretics, and β-blockers [3]. One of the oldest classes of antihypertensive drugs is CCBs, which are a heterogeneous group of medications [3]. Amlodipine (AML), a lipophilic, long-acting, third-generation dihydropyridine calcium channel blocker (CCB), has been widely used for the past 20 years. It is renowned for its efficacy and safety, supported by strong evidence from large randomized controlled trials demonstrating its effectiveness in reducing cardiovascular events [4]. AML works by preventing calcium from entering cardiac and vascular smooth muscle cells, thereby lowering peripheral vascular resistance [5]. It is prescribed to treat angina and high blood pressure, with several randomized studies confirming its effectiveness in managing angina pectoris. Due to its long half-life, AML is typically dosed once daily, which is advantageous for patient compliance [3].

Angiotensin II receptor antagonists (AIIRAs) have proven to be highly effective antihypertensive medications with superior tolerability since their introduction into clinical practice in 1995. When combined with thiazide diuretics and dihydropyridine CCBs, AIIRAs exhibit synergistic effects in lowering blood pressure without increasing adverse event incidence. Moreover, they have demonstrated benefits on mortality and morbidity in chronic renal disease and heart failure, particularly in the presence of type 2 diabetes [6]. The studied AIIRAs in this paper include Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS) and Irbesartan (IRB) are nonpeptide drugs that have been approved for use in the USA and Europe. A tetrazolo-biphenyl structure is shared by CAN, OLM, IRB and LOS, a benzimidazole group is shared by CAN and TLM [7].

For most hypertensive patients, concurrent administration of two or more medications is essential to manage blood pressure and reduce risk factors [2]. AML is often co-administered with AIIRAs to enhance their efficacy in patients with grade 1–2 hypertension [8]. Combining AIIRAs such as OLM, TLM, CAD, LOS, and IRB with CCBs like AML effectively lowers and regulates blood pressure (Fig. 1) [8, 9]. AIIRAs have an antisympathetic effect, which significantly improves AML's tolerance profile. They also reduce the likelihood of a heart rate increase with AML administration and partially mitigate the dose-limiting side effect of AML, which is peripheral edema [10].

AML and the studied AIIRAs have been analyzed using various methods, including spectrophotometry [11–14], spectrofluorometry [15–17] and square wave voltammetry [18–23] but these techniques are typically applied to dosage forms rather than biological fluids. Several chromatographic techniques have been developed for the determination of the studied drugs, including HPLC [24–26], GC-MS [27–29] and HPTLC [30–34]. Most of the reported methods for their determination in biological fluids depended on using HPLC [26, 35, 36] which uses large volume of expensive solvents. HPTLC is a widely used analytical technique due to its advantages, including low solvent usage, batch analysis, and rapid analysis. This technique allows for the concurrent measurement and identification of multiple compounds[37, 38] [39, 40].

None of the previously published methods employ HPTLC with UV detection suitable for the simultaneous determination of various AIIRAs and AML. Thus, there is a need to establish and validate a simple, rapid, and economical HPTLC method for their simultaneous determination in spiked plasma using reflectance/absorbance mode. For the first time, a new High-Performance Thin Layer Chromatography (HPTLC) system with UV detection had to be developed and tested for their simultaneous determination. In contrast to earlier studies that restricted use to pharmaceutical formulations, the present method's great sensitivity allowed for the identification of the compounds under study in plasma samples. The current process offers significant advantages in terms of saving time, solvents, effort, and cost, as five mixtures can be analyzed on the same plate with the same mobile phase. The proposed method was validated according to ICH standards.

2.1. Apparatus

A Camag-TLC system (CAMAG, Muttenz, Switzerland) with Scanner III was used for the chromatographic analysis. WinCATS version 1.4.4.6337 software was used to control the TLC. The radiation source used in the reflectance/absorbance mode was a high-pressure deuterium and halogen tungsten lamp. The scanning speed was set at 20 mm/s, and the scanner had a slit measuring 3 × 0.45 mm. Application of the sample on the plate was done using a Linomat V auto-sampler and a Hamilton syringe (100 µL, Bonaduz, Switzerland) with a mild nitrogen stream. Under rising conditions, the plate was developed in a twin-trough chamber with dimensions of 27.0 × 26.5 × 7.0 cm.

2.2. Chemicals and materials

Global Napi Pharmaceutical Co. kindly provided Amlodipine besylate, Candesartan cilexetil, and Losartan potassium (6th of October, Egypt). Sigma Pharmaceutical Company in Qewaisna, Egypt provided Telmisartan. Chemipharm Pharmaceutical Co. kindly supplied Olmesartan medoxomil (6th of October, Egypt). Irbesartan was graciously provided by Memphis Pharmaceutical Co. the (Cairo, Egypt). HPTLC measures were used to examine the pharmaceuticals under investigation for their purity, and the findings exceeded 98.00%. The following solvents (toluene, ethyl acetate, methanol, acetone, and acetic acid) were obtained from El-Nasr Pharmaceutical Chemicals (Abo-Zaabal, Cairo, Egypt).

2.3. Standard solution preparation

Stock solutions containing 1.0 mg/ml of the investigated medications (OLM, TLM, CAN, LOS, IRB, and AML) were made by putting 10 mg of each drug into 10 ml test tubes, sonicating the mixture for 10 minutes, and then adding more methanol to make the final volume of 10 ml. The examined AIIRAs were combined with AML. Next, working standard solutions (30, 50, 100, 140, 200, and 300 µg/ml) of the investigated AIIRAs medicines and (20, 40, 80, 120, 160, and 200 µg/ml) of AML were prepared by diluting stock solutions of each medication with methanol. Using a syringe, a 3 µL aliquot of the working solutions that had been created was applied to the silica gel plate, yielding final concentrations of (90, 150, 300, 420, 600, 900 ng/band) of the studied AIIRAs drugs and (60, 120, 240, 360, 480, 600 ng/band) of AML.

2.4. Plasma samples preparation

150 µL of plasma sample was spiked with 150 µL of a specific concentration of the medications under study (AIIRAs) and 150 µL of a specific concentration of AML standard solutions in an Eppendorf, Hamburg, Germany centrifuge tube. The mixture was then vortexed for two minutes. addition of 300 µL of acetonitrile, followed by a 5-minute vortex mix, and a 15-minute centrifugation at 14000 rpm at -4^o C. After extracting the clear supernatant from each centrifuge tube, the suggested methodology listed under the "Chromatographic conditions" was used to the analysis. Three distinct concentrations covering the linear range of each medication were used in six replicates for this technique.

2.5. Chromatographic conditions for the separation of the studied mixtures

The separation was performed on silica gel 60 F254 HPTLC plates (Merck, Darmstadt, Germany) with a stationary phase thickness of 200 µm and a particle size of 5 µm. The plates' exceptional purity and clarity were ensured by washing them with methanol prior to development. Plates were cut into 20 x 6 cm pieces with a solvent front at 5 cm. A mobile phase comprising 10 milliliters of Toluene: ethyl acetate: methanol: acetone: acetic acid (6: 1.5: 1: 0.5: 1, v/v/v/v/v) was saturated for 20 minutes at room temperature in order to separate the combinations under study. After development and drying, the plates were scanned at a specific wavelength for each mixture in a single run to detect both drugs. The detection wavelengths were set at 244 nm for the AML-OLM and AML-CAN mixtures, 247 nm for the AML-TLM mixture, and 254 nm for the AML-LOS and AML-IRB mixtures.

As hypertension is multifactorial disease, in recent years, there has been a marked increase in the use of combinations of antihypertensive drugs with complementary mechanisms of action. The combination of AML and the studied AIIRAs results in fully additive blood pressure reduction [41].

As all the previously documented HPTLC methods with UV detection were established for only one mixture and restricted to application in pharmaceuticals rather than plasma samples due to low sensitivity [30, 42, 43]. There is a pressing need to create a new HPTLC method with UV detection for the determination of the studied mixtures in spiked human plasma with acceptable levels of sensitivity. This current work provides a simple, quick, and sensitive method for simultaneous determination of AML and the studied AIIRAs "using UV detection" in spiked human plasma.

The use of mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6: 1.5: 1: 0.5: 1, v/v/v/v/v), gave well-separated, compacted and highly resolved bands for all the studied drugs, (0.50, 0.40, 0.62, 0.69, 0.73 ± 0.001 and 0.22 ± 0.002) for OLM, TLM, CAN, LOS, IRB and AML, respectively. Each mixture was detected in single run using one scanning wavelength as mentioned before. This is beneficial as it saves time, money, effort and solvents.

3.1. Method development

The studied AIIRA's exhibit a greater antihypertensive effect when taken in combination with AML [8, 9]. Unfortunately, the spectra of the studied drugs are overlapped with AML as shown in Fig. 2 for AML with CAN as a representative example. Numerous methods for the simultaneous determination of AML with the studied AIIRA drugs using derivative spectrophotometry or ratio derivatives have been reported [11, 13, 14]. However, these methods have significant drawbacks, such as low signal, poor sensitivity, an inconvenient signal-to-noise ratio, poor robustness, and susceptibility to potential interferences from excipients[44–46]. HPTLC technique allows simultaneous determination of the mixed drugs at one maximum wavelength without any interference from each other or from the combined excipients as they are separated at different R_f values[47–50]. The current study used single detection wavelength for each mixture of the studied five mixtures utilizing the same chromatographic system (stationary and mobile phases) for the separation and simultaneous detection and quantification of the studied AIIRAs and AML.

Several trials were carried out to select the most suitable mobile phase for simultaneous determination of AML with each of the studied AIIRAs drugs. It was found that mixture of dichloromethane, methanol and acetic acid move the studied drugs to higher R_f values near the solvent front. On the other hand, combination between Toluene with methanol and acetic acid gives poor resolution between AML with all the studied AIIRAs drugs. Also, mixture of Toluene, ethyl acetate, methanol, acetonitrile and acetic acid produce the spot of AML with tailing. So, mixture of Toluene: ethyl acetate: methanol: acetone: acetic acid (6: 1.5: 1: 0.5: 1, v/v/v/v/v) were used. The selected mobile phase should be able to give compact spots of the drugs with suitable R_f values. The obtained Rf values for the studied drugs are (0.50, 0.40, 0.62, 0.69, 0.73 ± 0.001 and 0.22 ± 0.002) for OLM, TLM, CAN, LOS, IRB and AML respectively. Figure 3 shows HPTLC densitogram of AML and CAN using Toluene: ethyl acetate: methanol: acetone: acetic acid (6: 1.5: 1: 0.5: 1, v/v/v/v/v) as a mobile phase.

3.2. Validation of the Method

The developed method was fully validated according to the ICH guidelines and complied for linearity, accuracy, and precision, detection and quantification limits, and robustness. The statistical analysis was carried out with the 95% confidence level.

3.2.1. Linearity

Six concentration levels (three replicates for each) for all of the studied drugs were selected to construct the calibration curves. Calibration parameters are given in (Table 1) showing high correlation from the calculated correlation coefficients (r) between the investigated concentrations and the corresponding obtained peak areas. They were in the range 0.9939–0.9998, expressing good linear relationship of the suggested method. Figure 4 shows three dimensional densitogram for mixture of AML and CAN as a representative example over the ranges of (60–600 ng/ band) and (90–900 ng/band) respectively.

Table 1

Statistical data for the simultaneous HPTLC determination of some AIIRAs – Amlodipine mixtures with UV detection
Parameters	OLM - AML		TLM – AML		CAN - AML		LOS - AML		IRB - AML
Parameters	OLM	AML	TLM	AML	CAN	OLM	AML	TLM	AML	CAN
Linear range (ng/band)	90–900	60–600	90–900	60–600	90–900	60–600	90–420	60–480	90–480	60–480
Correlation coefficient, r	0.9984	0.9939	0.9998	0.9986	0.9992	0.9992	0.9968	0.9971	0.9993	0.9984
Determination Coefficient, r2	0.9969	0.9880	0.9996	0.9972	0.9984	0.9985	0.9937	0.9942	0.9986	0.9968
Slope ± SD (a)	6.66 ± 0.18	5.81 ± 0.32	5.44 ± 0.05	5.75 ± 0.15	2.92 ± 0.06	3.32 ± 0.06	4.23 ± 0.15	2.27 ± 0.07	6.86 ± 0.11	2.15 ± 0.05
Intercept ± SD (a)	414.35 ± 48.93	-74.61 ± 25.28	7.02 ± 26.90	− 249.10 ± 21.77	1224.48 ± 20.42	87.96 ± 13.37	397.03 ± 27.71	134.85 ± 9.70	80.82 ± 34.13	43.56 ± 10.40
RF	0.50 ± 0.001	0.20 ± 0.002	0.41 ± 0.001	0.20 ± 0.002	0.61 ± 0.001	0.20 ± 0.002	0.62 ± 0.001	0.20 ± 0.002	0.76 ± 0.001	0.20 ± 0.002
LOD (ng/band)	24.26	14.36	16.32	12.49	23.12	13.29	21.62	14.10	16.42	15.96
LOQ (ng/band)	73.52	43.52	49.45	37.86	70.05	40.28	65.50	42.73	49.76	48.37
(a) Average of three determinations

3.2.2. Limits of detection and quantification

The calculated LOD and LOQ for the studied drugs using the proposed method indicated high sensitivity (Table 1). They are calculated from the standard deviation of the intercepts response and the slope of the calibration curve using these equations: LOD = 3 Ϭ/ b and LOQ = 10 Ϭ/ b, where b denotes the slope of the calibration curve and Ϭ denotes the standard deviation of the intercept.

The calculated LOD and LOQ values of the proposed method with UV detection were found to be, respectively, (14.36, 24.26 ng/band) and (43.52, 73.52 ng/band) for the AML – OLM mixture, (12.49, 16.32 ng/band) and (37.86, 49.45ng/band) for the AML – TLM mixture, (13.29, 23.12 ng/band) and (40.28, 70.05 ng/band) for the AML – CAN mixture, (14.10, 21.62 ng/band) and (42.73, 65.50 ng/band) for the AML – LOS mixture, and (15.96, 16.42 ng/band) and (48.37, 49.76 ng/band) for the AML – IRB mixture.

3.2.3. Accuracy

The results obtained in (Table 2) by carrying out six times measurements of three different concentrations of each drug were satisfactory as indicated, the obtained recoveries ranged from 97.92 to 103.76 which indicated good accuracy of the suggested method. As the proposed method's great accuracy was judged from the closeness of the recovery percentages to 100% with low values of standard deviation.

Table 2

Evaluation of the accuracy of the proposed HPTLC method with UV detection for simultaneous determination of the studied AIIRAs– Amlodipine mixtures
Studied mixtures	AIIRAs OLM, TLM, CAN, LOS, IRB			AML
Studied mixtures	Conc. (ng\band)	Amount found (ng/band)	% Recovery (a) ± SD	Conc. (ng\band)	Amount found (ng/band)	% Recovery (a) ± SD
OLM - AML	150	146.88	97.92 ± 1.06	120	120.44	100.36 ± 2.33
	300	302.23	100.74 ± 1.91	240	236.12	98.38 ± 1.56
	600	602.56	100.43 ± 0.99	480	472.65	98.47 ± 1.09
TLM – AML	150	152.20	101.47 ± 2.16	120	118.97	99.15 ± 1.14
	420	417.09	99.31 ± 0.89	360	354.80	98.56 ± 2.08
	900	896.69	99.63 ± 1.05	600	608.89	101.48 ± 0.99
CAN - AML	150	147.01	98.00 ± 2.57	120	118.76	98.96 ± 1.57
	420	430.37	102.47 ± 1.82	360	358.96	99.71 ± 2.01
	900	904.12	100.46 ± 2.02	600	600.67	100.11 ± 2.47
LOS - AML	120	119.85	99.87 ± 2.60	90	92.01	102.24 ± 2.54
	270	276.11	102.26 ± 0.66	270	272.39	100.89 ± 2.19
	420	411.81	98.05 ± 1.13	480	486.65	101.38 ± 1.51
IRB - AML	120	124.52	103.76 ± 2.44	90	91.37	101.52 ± 2.83
	270	275.97	102.21 ± 2.58	270	274.16	101.54 ± 2.03
	420	424.22	101.01 ± 1.77	420	416.02	99.05 ± 1.17
(a) Average of six determinations at each concentration level

3.2.4. Precision

Intra and inter-day precision were expressed as percent relative standard deviation (% RSD) the results were given in (Table 3). For all concentration levels, the RSD didn't exceed 2.85% indicating good precision of the proposed method (Fig. 5) they assessed by analyzing six replicates’ measurements of three different concentrations of each drug intra-daily and in three distinct days.

Table 3

Precision of the proposed HPTLC method with UV detection for simultaneous determination of the studied AIIRAs– Amlodipine mixtures at the intra- and inter-day levels
Studied mixtures	AIIRAs OLM, TLM, CAN, LOS, IRB			AML
Studied mixtures	Conc. (ng\band)	Intra-day RSD %(a)	Inter-day RSD %(b)	Conc. (ng\band)	Intra-day RSD %(a)	Inter-day RSD %(b)
OLM - AML	150	2.29	1.69	120	0.99	2.35
	300	0.69	1.30	240	1.71	1.26
	600	1.13	0.96	480	1.17	1.39
TLM – AML	150	2.02	2.34	120	2.55	2.21
	420	0.90	1.55	360	2.77	2.10
	900	1.04	1.27	600	2.64	1.30
CAN - AML	150	2.40	2.23	120	1.46	2.39
	420	1.18	2.26	360	2.26	1.89
	900	2.06	2.23	600	2.14	1.70
LOS - AML	120	0.98	1.12	90	2.09	2.25
	270	2.07	2.39	270	1.61	1.99
	420	1.11	1.09	480	1.59	1.88
IRB - AML	120	1.88	2.34	90	2.80	2.83
	270	1.27	2.85	270	2.22	2.00
	420	1.55	1.28	420	1.37	2.46
(a) Average of 6 determinations at each concentration level.
(b)Average of 18 determinations at each concentration level over three distinct days.

3.2.5. Robustness

A small variation in the proposed method parameter such as composition of the mobile phase, scanning wavelength, and saturation time, it was found that there was no significant effect on the method performance as given in (Table 4) so the method is considered to be robust.

Table 4

Results for the investigation of the robustness of the proposed HPTLC method with UV detection for simultaneous determination of the studied AIIRAs– Amlodipine mixtures
Parameters	OLM - AML		TLM – AML		CAN - AML		LOS – AML		IRB - AML
Parameters	OLM	AML	TLM	AML	CAN	OLM	AML	TLM	AML	CAN
Optimum parameters	100.43 ± 0.99	100.36 ± 2.33	101.47 ± 2.16	99.15 ± 1.14	98.00 ± 2.57	98.96 ± 1.57	102.26 ± 0.66	100.89 ± 2.19	101.01 ± 1.77	99.05 ± 1.17
Composition of the mobile phase Toluene: ethyl acetate: methanol: acetone: acetic acid (5.8: 1.7: 1: 0.5: 1, v/v/v/v/v)	98.13 ± 1.15	99.26 ± 2.56	101.52 ± 1.22	100.67 ± 1.37	97.82 ± 2.28	98.44 ± 1.08	99.10 ± 0.89	101.35 ± 2.30	102.07 ± 1.49	100.73 ± 1.86
Toluene: ethyl acetate: methanol: acetone: acetic acid (5.8: 1.5: 1.1: 0.5: 1.1, v/v/v/v/v)	98.71 ± 2.13	100.53 ± 1.25	101.54 ± 1.78	99.43 ± 0.69	98.60 ± 1.70	101.71 ± 1.46	102.36 ± 1.38	99.02 ± 2.47	97.30 ± 2.36	101.41 ± 1.57
Toluene: ethyl acetate: methanol: acetone: acetic acid (6: 1.5: 0.8: 0.7: 1, v/v/v/v/v)	102.33 ± 1.70	101.47 ± 1.52	98.70 ± 2.53	101.45 ± 1.36	99.17 ± 0.79	98.53 ± 1.22	100.78 ± 1.59	102.71 ± 2.46	99.60 ± 1.58	98.15 ± 1.47
Scanning wavelength, 242 246	101.44 ± 1.56 98.25 ± 2.17	101.18 ± 1.44 99.38 ± 1.30			99.47 ± 1.82 101.26 ± 1.57	100.39 ± 1.68 98.36 ± 2.26
245 249			97.56 ± 1.66 99.34 ± 2.10	99.46 ± 1.36 100.57 ± 1.48
252 256							102.61 ± 1.50 101.83 ± 1.56	98.36 ± 1.77 97.91 ± 0.78	100.91 ± 2.34 102.15 ± 2.41	101.26 ± 1.89 100.77 ± 1.76
Saturation time 18 mins 22 mins	98.56 ± 1.44 100.48 ± 2.18	101.61 ± 1.40 102.51 ± 1.97	100.72 ± 2.23 99.37 ± 1.74	102.17 ± 0.86 99.50 ± 1.59	101.66 ± 1.57 98.46 ± 1.63	100.83 ± 1.19 101.49 ± 2.16	97.60 ± 1.99 100.85 ± 1.71	100.49 ± 2.16 101.53 ± 1.48	101.67 ± 1.91 101.49 ± 1.57	98.77 ± 1.43 97.43 ± 1.75
(a) Average of six determinations

3.3. Application of the proposed HPTLC method in spiked human plasma

The proposed method was successfully used for the determination of the studied drugs in spiked human plasma. Six replicate measurements were made of three distinct concentrations of each drug utilizing the chromatographic conditions mentioned before. Since it was difficult to get blood from hypertensive patients who had received the examined medicines, we decided to test our suggested method on samples of spiked human plasma to see if it could detect the studied drugs' residual levels in human plasma after delivery. Blank plasma sample without spiking with the studied drugs was analyzed as shown in (Fig. 6a). Then, the plasma samples were spiked with the studied drug mixture at 3 different concentration levels for each drug as illustrated in (Fig. 6b). The high estimated mean percent recovery indicates that the existing methodology for examining these antihypertensive medications in spiked plasma is effective and reliable (Table 5). The recovery percentages ranged from 93.31 to 107.92 and standard deviations were in the range of (0.82–2.54).

Table 5

Application of the proposed HPTLC method with UV detection for simultaneous determination of the studied AIIRAs– Amlodipine mixtures in spiked human plasma
Studied mixtures	AIIRAs OLM, TLM, CAN, LOS, IRB			AML
Studied mixtures	Conc. (ng\band)	Amount found (ng/band)	% Recovery (a) ± SD	Conc. (ng\band)	Amount found (ng/band)	% Recovery (a) ± SD
OLM - AML	150	144.19	96.13 ± 2.13	120	126.69	105.57 ± 1.20
	300	316.20	105.40 ± 1.70	240	248.01	103.34 ± 2.61
	420	411.49	97.97 ± 1.43	360	369.59	102.66 ± 1.81
TLM – AML	150	142.28	94.85 ± 1.18	120	127.17	105.98 ± 2.40
	420	404.13	96.22 ± 1.92	360	349.35	97.04 ± 1.16
	600	579.98	96.66 ± 2.54	480	458.44	95.51 ± 0.89
CAN - AML	150	157.64	105.09 ± 0.82	120	112.97	94.14 ± 1.44
	300	282.11	94.04 ± 1.79	360	339.60	94.33 ± 2.37
	420	437.67	104.21 ± 1.50	600	563.38	93.90 ± 1.98
LOS - AML	120	126.24	105.20 ± 1.73	90	83.98	93.31 ± 2.16
	270	282.08	104.47 ± 1.12	270	256.85	95.13 ± 0.91
	420	447.10	106.45 ± 2.53	420	404.65	96.35 ± 2.37
IRB - AML	120	114.48	95.40 ± 1.06	90	97.13	107.92 ± 1.45
	210	219.36	104.46 ± 1.38	210	216.92	103.29 ± 1.96
	360	345.85	96.07 ± 2.11	360	378.32	105.09 ± 2.53
(a) Average of six determinations at each concentration level

The proposed high-performance thin-layer chromatographic (HPTLC) method with UV detection provides a quick, user-friendly, cost-effective, and sensitive approach for the simultaneous determination of Amlodipine (AML) and the studied Angiotensin II Receptor Antagonists (AIIRAs). This method allows for the detection of both drugs in a single run at one scanning wavelength. Notably, there are no published HPTLC methods that simultaneously determine AML with various AIIRA members. Due to the high sensitivity, simplicity, and selectivity of the HPTLC method, this approach successfully analyzed AML and the studied AIIRAs in human plasma without interference from plasma constituents. This methodology can be applied in future pharmacokinetic studies and can effectively determine the plasma concentrations of the studied drugs, owing to the excellent recovery of the drugs from plasma.

Ethics approval and consent to participate:

Not applicable.

Consent for publication:

Not applicable.

Competing interests:

All authors confrm that there are no competing interests.

Funding:

Open access funding provided by The Science Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). There is no funding to declare

Author Contribution

Ahmed A. Khorshed: Conceptualization, Methodology, Investigation, Data curation, Formal analysis, Visualization, Writing – original draft. Fatma M. Abdelnaeem: Methodology, Investigation, Conceptualization, Data curation, Formal analysis Writing – original draft. Dalia M Nagy: Conceptualization. Mohamed Oraby: Investigation, Writing – review & editing. Sayed M. Derayea: Writing – review & editing.

Acknowledgements:

Not applicable

Availability of data and materials:

All data generated or analyzed during this study are included in this published article.

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High-Performance Thin-Layer Chromatographic method for Simultaneous Determination of Some Angiotensin II Receptor Antagonists with Amlodipine in spiked human plasma with UV Detection

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Abstract

Figures

1. Introduction

2. Experimental

2.1. Apparatus

2.2. Chemicals and materials

2.3. Standard solution preparation

2.4. Plasma samples preparation

2.5. Chromatographic conditions for the separation of the studied mixtures

3. Results and discussion

3.1. Method development

3.2. Validation of the Method

3.2.1. Linearity

3.2.2. Limits of detection and quantification

3.2.3. Accuracy

3.2.4. Precision

3.2.5. Robustness

3.3. Application of the proposed HPTLC method in spiked human plasma

Conclusion

Declarations

Funding:

Author Contribution

Acknowledgements:

Availability of data and materials:

References

Additional Declarations

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Version 1