Development of a RP-HPLC method for the simultaneous analysis of diltiazem and statin: Application in pharmaceuticals and human serum

Abstract

High-performance liquid chromatographic (HPLC) method has been developed and validated for the simultaneous determination of diltiazem and statins in raw materials, their pharmaceutical formulations and human serum. In HPLC, diltiazem and statins are chromatographed using acetonitrile–water (85 : 15 v/v, pH 2.6 ± 0.02) as the mobile phase at a flow rate of 1.0 mL min⁻¹ at ambient temperature. The separation is carried out on a Hiber®, 250-4.6 RP-18 column, equipped with a UV/visible detector at 230 nm. All the statins eluted at a different retention time and each showed good resolution from diltiazem. The method has been successfully applied to pharmaceutical formulations because no chromatographic interferences from the tablet excipients are found. The linearity is found to be in the range 0.625–20 μg mL⁻¹. The suitability of the method for the quantitative determination of the drugs is proven by validation in accordance with the requirements laid down by the International Conference on Harmonization (ICH) guidelines. The validation results, together with statistical treatment of the data, demonstrated the reliability of this method.

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Introduction

Hypertension and hyperlipidemia are correlated to each other and have an additional effect on coronary heart disease and the associated mortality rate, since cardiovascular disease is closely related to factors such as hypertension, high cholesterol levels or diabetes. These factors are related to family history, sex, age, obesity and diabetes.^1–10 Coadministration of antihypertensive, hypolipemiant and antidiabetic drugs is frequently used in treatment.^11–14 One of the most used combinations consists of a synergic association of a diuretic (chlorthalidone, hydrochlorothiazide, etc.) and an angiotensin II receptor antagonist (valsartan, telmisartan, etc.) to control the hypertension, with a statin (fluvastatin, simvastatin, etc.) to reduce the cholesterol levels.¹⁵ Therefore, it has been hypothesized that coadministration of an antihypertensive agent and statin might be an effective therapeutic option for multiple cardiovascular risk factors and concomitant management of elevations in blood pressure and LDL cholesterol.^16–21

Diltiazem is a peripheral and coronary vasodilator with limited negative inotropic activity inhibiting cardiac conduction, particularly at the sino-atrial and atrioventricular nodes. It is available for intravenous administration in the treatment of various cardiac arrhythmias (atrial fibrillation or flutter and paroxysmal supraventicular tacchycardia) and Raynaud's syndrome.^22,23 The statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are the most effective among all hypolipidemic agents.²⁴ These agents are highly effectual in enhancing the HDL levels while, reducing LDL cholesterol, overall cholesterol, apolipoprotein B and triglyceride levels.²⁵ In addition, statins may improve the vasodilatation capacity of large arteries and may thus contribute to BP lowering in patients treated with both an anti-hypertensive and a statin.²⁶ Literature survey revealed that number of assay methods have been used for analysis of diltiazem in bulk drug, pharmaceutical preparations and serum using different techniques including colorimetry,²⁷ stripping voltammetry and flow amperometry,²⁸ Raman spectroscopy,²⁹ capillary electrophoresis ³⁰, polarimetry,³¹ spectrophotometry³² and RP-HPLC.^33–35 However, several analytical procedures have also been described for simultaneous separation and enantio-separation of diltiazem, its analogs, possible degradation products and metabolites.^33,36,37 A number of assays have been reported for the quantitative determination of statins in pharmaceutical dosage forms, bulk material and human plasma or serum. Nearly all these assay methods are based on either HPLC or GC techniques³⁸ like assay scheme for rosuvastatin,³⁹ simvastatin^40,41 and atorvastatin.⁴² An HPLC method for simultaneous determination of HMG-CoA reductase inhibitors in pharmaceutical formulations and plasma or serum has been reported.^39,43,44

There are very few analytical methods developed for the simultaneous determination of a combination of different kind of drugs used in treatment (antihypertensive, hypolipemiant, antidiabetic, antithrombotic). These methods have been applied to pharmaceuticals and plasma samples.^45–47 The present study was conducted to analyze the diltiazem and statins in pharmaceutical formulations and human serum. We present the development and validation of a HPLC assay using UV detector for measurement and allowed determination of each agent without the need for development of separate and distinct methods for each analyte.

Experimental

Instrumentation

A Shimadzu HPLC system equipped with LC-10 AT VP pump and SPD-10 A VP UV-VIS detector was utilized. The chromatographic system was integrated via Shimadzu model CBM-102 to P-IV computer loaded with Shimadzu CLASS-VP software (Version 5.03) for data acquisition and mathematical calculations. Rheodyne manual injector fitted with a 20 μL loop, a Hiber®, RT 250-4.6 Purospher® STAR RP-18 column and DGU-14 AM on-line degasser and Mettler Toledo electronic balance, microlitre syringe and micropore filtration assembly were used in this study.

Materials and reagents

Diltiazem, was gifted from Hilton Pharma (Private) Limited. Rosuvastatin (X-plended 20 mg), atorvastatin (Atopitar 10 mg) and simvastatin (Atcol 10 mg) Pharm Evo (Pvt.) Ltd., Atco Pharma(Pvt.) Ltd., and Geofman Pharma (Pvt.) Ltd., respectively, were purchased locally. Reference standards of all these statins were supplied by lab-9 of the Department of Chemistry at the University of Karachi. Each product was labelled and had an expiry date of not less than 365 days at the time of study. All reagents used were of HPLC grade. Methanol and phosphoric acid 85% (Merck, Germany) and HPLC grade deionized filtered water were used to prepare the mobile phase. Stock solutions of diltiazem and statins were prepared in the mobile phase. Fresh working solutions were prepared daily. All solutions were filtered (0.45 μm) and degassed using a sonicator.

Preparation of solutions

Standard solutions of diltiazem and each statin were prepared by dissolving appropriate amounts of drug in mobile phase acetonitrile–water (85 : 15, v/v) to yield final concentrations of 100 μg mL⁻¹. Seven concentrations of each drug were prepared by making serial dilutions from stock solutions. For the assay preparation, the content of 20 tablets were powdered, a weighed portion of the powder equivalent to a suitable amount of drug (according to the labeled claims) was transferred into a 50 mL volumetric flask. The drug was fully dissolved in mobile phase and then diluted with this solvent up to the mark, seven dilutions of each drug were prepared, a portion of this solution was filtered through a disposable 0.45 μm filter and then injected.

Serum drug solution

Blood samples were collected from healthy volunteers and then centrifuged at 3000 rpm for 10 min. The supernatant obtained was stored at −20 °C. After thawing, serum was deprotonated by acetonitrile and spiked daily with working solutions to produce desired concentrations of diltiazem and statins. A 20 μL volume of each sample was injected and chromatographed under the above conditions.

Chromatographic conditions

The chromatographic analysis was performed at ambient temperature with isocratic elution. The mobile phase consisted of acetonitrile–water (85 : 15 v/v) with pH adjusted to 2.6 ± 0.02 with phosphoric acid (85%) and pump at a flow rate of 1 mL min⁻¹, sample volume of 20 μL was injected in triplicate onto the HPLC column and effluents were screened over the dative UV region at 230 nm.

Results and discussion

The primary target in developing this LC method was to achieve simultaneous determination of these drugs in pharmaceutical formulations and also in human serum under common conditions that are applicable for routine quality control, research and development of these drugs in ordinary laboratories. To the best of our knowledge no method is available in literature which can analyze these drugs simultaneously, so there was a need to develop a method which could simultaneously determine all these drugs.

The development of HPLC methods for the determination of drugs has received considerable attention in recent years because of their importance in the quality control of drugs and drug products.

This work was intended to develop a precise, reliable, and least time consuming method, based on reverse-phase HPLC separation combined with UV detection, for simultaneous drug assay in raw materials, bulk drug samples, dosage formulations and, especially, in human serum.

Method development

Method development is very important for drug quality control, stability, metabolism, pharmacokinetics and toxicity studies. Simple, efficient and economical analytical methods are very critical requirements for all these types of investigation.⁴⁸

Method optimization and isocratic scouting. In order to develop an efficient method for the analysis of these analytes simultaneously, preliminary tests were performed with the purpose of selecting the best and optimum conditions. Parameters, such as detection wavelength, mobile phase proportions, optimum pH and concentration of the standard solutions were exhaustively studied. Mobile phase was selected in terms of its components and proportions. Different ratios of acetonitrile (ACN) and water (90 : 10, 80 : 20, 70 : 30, 60 : 40, 50 : 50, 40 : 60, 30 : 70, 20 : 80 and 10 : 90) as well as methanol and water were tried.

An HPLC-UV method was developed and successfully validated for the simultaneous quantitation of diltiazem and statins. Chromatographic parameters were isocratically optimized on a set of different C18 stationary phases with different lengths and particle sizes. Isocratic scouting does not involve re-equilibration of the column.⁴⁹ First, a Hypersil, ODS, C18 (150 × 4.6 mm, 5 μm) column was experimented with a variety of mobile phases but the consequences still were not adequate. Analyte peaks were not efficient, reproducible with short analyses time (<10 min) and acceptable resolution, because of peak tailing and placebo obstruction in the formulations. Best choice of the stationary phase that provided satisfactory peak shape, resolution and run time was based on a Hiber®, RT 250-4.6 Purospher® STAR RP-18 endcapped (5 μm). A variety of mobile phases were investigated for optimization of chromatographic conditions with respect to the quantity and different rates of organic modifier. In order to select an appropriate organic modifier, different compositions of acetonitrile and methanol were experienced. Beneficial partition was established with acetonitrile. Various mobile phase compositions were attempted to separate diltiazem and statins and the amount of acetonitrile varied, when it was less than 65% of the total volume of mobile phase diltiazem was retained on the column. Mobile phase between 65–82% of acetonitrile showed that statins could not be separated but each showed a good resolution from diltiazem. The optimum results (capacity factor (k′), specificity, good resolution and short omega peak) were obtained when the copmposition of acetonitrile in the mobile phase was 85%. The fitness of the mobile phase was achieved on the foundation of the sensitivity, suitability for stability studies, run time required for the investigation, simplicity of preparation, and utilization of readily available cost-effective solvents. Variation in pH of the mobile phase had a great influence on the retention time, chromatographic peak and efficiency of the chromatographic system. pH of the mobile phase varied between 2.5 to 3.5 and best results were attained at pH 2.6 ± 0.02, if the pH is less than 2.6 peak tailing was observed while at a higher pH the diltiazem and statins peak distorted and retention times were higher. For detection wavelength, UV spectra of both diltiazem and statins overlapped. It was found that both the drugs exhibited the highest response (maximum UV absorption) at 230 nm and offered a greatest intensity with smallest interference. A typical chromatogram of the separation of all the drugs is shown in Fig. 1.

Fig. 1 Representative chromatogram showing resolution between diltiazem and statins in a reference standard.

Peak identification. Peak of diltiazem, rosuvastatin, atorvastatin and simvastatin were identified using retention times by matching their values with those of standards as eluted out at 3.09, 5.7, 6.7 9.3 min, respectively.

Method validation

Method validation has received considerable attention in the literature, by industrial committees and regulatory agencies.⁵⁰ The developed method was validated according to the ICH guidelines⁵¹ under such parameters as system suitability, selectivity, specificity, linearity, accuracy, precision and sensitivity (detection and quantification limit).

System suitability test. Before sample analysis, six consecutively replicate analysis of the drug at a concentration of 50 μg mL⁻¹ for both diltiazem and statins were assessed in order to investigate the suitability parameters including repeatability, retention factor, peaks symmetry, column efficiency (theoretical plates) and resolution. Results are depicted in Table 1, sharp and symmetrical peaks were obtained with good baseline resolution (Fig. 1) and tailing, % RSD of different parameters are shown in Table 1.

Table 1 System suitability parameters^a

Parameters	% RSD
	DLZ	ROS	ATR	SIM
a Diltiazem; DLZ, Rosuvastatin; ROS, Atorvastatin; ATR, Simvastatin; SIM.
Retention time (Rt in minutes)	0.1846	0.3497	0.4859	0.3783
Capacity factors (K′)	0.1851	0.3492	0.4856	0.3786
Theoretical plates/N	1.3241	0.0440	0.5192	0.0385
Tailing factor (T)	1.0247	1.5655	1.5890	1.9241
Resolution (R)	0.6251	0.4417	2.5923	2.7442

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Tailing (symmetry) factor (T) and number of theoretical plates (N) was calculated by formulas: T = W/(2Wa) and N = 5.545(t_R/W_1/2)², where W is the peak width at 5% height from baseline, Wa the peak front edge width at the same height and W_1/2 is the peak width at half height. According to data of a robustness test study we proposed criteria for system suitability test (tailing factors <2, numbers of theoretical plates >4000 and repeatability (RSD) of five replicate samples to be not more than 1.5, (peak area for replicate analysis). The robustness test is used to verify that the resolution and repeatability of the system are adequate for the analysis intended.

Selectivity/Specificity. Specificity/selectivity were set up by a separate chromatographic analysis of the excipient mixtures. No interfering peaks of excipients, present in the formulations were observed (Fig. 2), and provides evidence that no interfering peaks occur in the region where peaks of diltiazem and statins are eluted.

Fig. 2 Representative chromatogram showing resolution between diltiazem and statins in pharmaceutical formulations.

Specificity was also determined by screening different samples of controlled human serum, which were free from interfering endogenous plasma components. This is evidenced by the lack of interfering peaks in the chromatograms of serum samples. Fig. 3 represents a typical chromatogram of human serum containing all drugs analysed. A typical chromatogram of blank serum is shown in Fig. 4. The method demonstrated good resolution between the peaks and was found to be free of interferences.

Fig. 3 Representative chromatogram showing resolution between diltiazem and statins in human serum.

Fig. 4 A representative chromatogram of blank serum.

Linearity. Linearity of the proposed method was investigated by analyzing seven dilutions of both (diltiazem and statins) in the range 0.625–20 μg mL⁻¹. The linear regression analysis results (mean values) are given in Table 2. The calibration curves show excellent linearity with a good fit (R²) > 0.999, an apparent a linear affiliation between concentration and peak área.

Table 2 Regression statistics and sensitivity (μgmL⁻¹) of the proposed method^a

Analytes	Goodness of fit (R^2)	Standard error	Standard error of estimate	Intercept	Slope	LOD	LOQ
a Diltiazem; DLZ, Rosuvastatin; ROS, Atorvastatin; ATR, Simvastatin; SIM.
Bulk material (0.625–20 μg mL^−1)
DLZ	0.9997	2417	4256.05	148112	25454	0.0029	0.0089
ROS	0.9997	3002	5287.49	83864	27214	0.0094	0.0286
ATR	0.9997	2641	4651.52	71340	23788	0.0017	0.0053
SIM	0.9995	1366	2405.85	15723	9934	0.0042	0.0127
Serum (0.625–20 μg mL^−1)
DLZ	0.9996	2424	4264	148105	25467	0.0031	0.0093
ROS	0.9994	3018	5281	83859	27211	0.0098	0.0345
ATR	0.9995	2657	4624	71389	23752	0.0025	0.0064
SIM	0.9995	1359	2412	15716	9945	0.0051	0.0138

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Accuracy and recovery. Method accuracy was evaluated by measuring the recovery of known amounts of each drug which were spiked into their corresponding formulation. These spiked matrices were expressed as a percentage at three concentration levels 80% (n = 3), 100% (n = 6) and 120% (n = 3). Accuracy results show that the recovery values in bulk drug and serum were in the range of 97.9–102% (Table 3), which was well within the acceptable boundary.

Table 3 Method accuracy from recovery assays for the studied analytes^a

% Recovery					Recovered conc. μg mL^−1
Conc. μg mL^−1	DLZ	ROS	ATR	SIM	DLZ	ROS	ATR	SIM
a 80% = 12 μgmL^−1; 100% = 15 μgmL^−1; 120% = 18 μgmL^−1, n = No of sample injected. Diltiazem; DLZ, Rosuvastatin; ROS, Atorvastatin; ATR, Simvastatin; SIM.
0.625	99.99	100.0	99.98	100.09	0.62	0.62	0.62	0.62
1.25	100.0	99.65	99.99	99.87	1.25	1.24	1.24	1.24
2.5	100.0	100.59	100.0	100.15	2.50	2.51	2.500	2.50
5	99.9	99.75	99.92	99.97	4.99	4.98	4.99	4.99
10	100.07	100.07	99.79	99.98	10.00	10.00	9.97	9.99
15	99.99	100.07	99.71	99.99	14.99	15.01	14.95	14.99
20	99.94	99.95	100.87	100.03	19.98	19.99	20.17	20.00
Spiking (bulk material % level)
80	99.80	101.5	99.96	100.21	11.97	12.18	11.99	12.02
100	100.19	100.11	100.18	102.15	15.02	15.01	15.02	15.32
120	100.17	100.14	99.97	100.53	18.03	18.02	17.99	18.09
Spiking (serum % level)
80	98.95	101.78	97.99	99.97	11.87	12.21	11.75	11.99
100	98.12	100.01	99.45	101.54	14.71	15.00	14.91	15.23
120	99.01	100.75	98.65	101.04	17.82	18.13	17.75	18.18

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Recoveries of different concentrations of all the drugs from pharmaceutical formulations and serum were calculated by dividing the integrated peak area by the respective nominal drug concentration for calibration curve and serum samples, and expressed as the recovery (%).

Precision. The intra- and inter-day variations (method precision) were analyzed by coefficient of variation (CV) throughout the linear range of concentrations. The precision articulated as inter- and intra-day CV are listed in Table 4, indicating that there was no significant difference for the assay which was tested within day and between days which indicates that the proposed method is precise and reproducible.

Table 4 Intra- and inter-day, variations/intermediate precision of the method^a

Day 1 (CV)					Day 2 (CV)
Conc. μg mL^−1	DLZ	ROS	ATR	SIM	DLZ	ROS	ATR	SIM
a Diltiazem; DLZ, Rosuvastatin; ROS, Atorvastatin; ATR, Simvastatin; SIM, Cofficient of variation; CV.
0.625	0.13	0.10	0.58	0.24	0.65	0.85	0.93	0.95
1.25	0.22	0.63	0.12	0.183	0.46	0.79	0.13	0.32
2.5	0.67	0.78	0.48	0.159	0.67	0.84	0.12	0.35
5	0.88	0.55	0.29	0.96	0.13	0.97	0.56	0.65
10	0.69	0.92	0.65	0.176	0.59	0.86	0.16	0.59
15	0.27	0.65	0.64	0.89	0.33	0.14	0.28	0.22
20	0.83	0.57	0.48	0.28	0.61	0.19	0.48	0.69
Serum/μg mL^−1
0.625	0.18	0.18	0.49	0.96	0.98	0.65	0.76	0.86
1.25	0.35	0.59	0.16	0.112	0.99	0.78	0.69	0.93
2.5	0.68	0.68	0.65	0.161	0.97	0.58	0.98	0.89
5	0.73	0.64	0.38	0.98	0.67	0.97	0.101	0.97
10	0.37	0.39	0.28	0.13	0.58	0.68	0.113	0.94
15	0.74	0.94	0.45	0.143	0.98	0.84	0.98	0.92
20	0.75	0.67	0.39	0.99	0.97	0.98	0.89	0.99

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Detection and quantification limit. The analytical sensitivity of the method was anticipated from the signal to noise ratio 3 : 1 and 10 : 1 summarized in Table 2. The minimum limits at which the analytes could be readily detected and quantified for diltiazem, rosuvastatin, simvastatin and atorvastatin were 0.0029, 0.0094, 0.0017, 0.0042 μg mL⁻¹ and 0.0089, 0.0286, 0.0053, 0.0127 μg mL⁻¹ respectively, which suggested that a nanogram quantity of each drug can be estimated accurately.

Ruggedness. The ruggedness (degree of reproducibility) of the method can be obtained under a variety of conditions, such as different laboratories, analysts, instruments, environmental conditions, operators, materials and different days.^52,53 The assay results indicated that the method was capable with high precision (Table 4). Results of CV for inter- and intra-day reproducibility of two different days prove the ruggedness of developed method.

Robustness. Robustness of the method was executed by designed modifications made to the method parameters^52,53 such as composition, flow rate, pH of the mobile phase, detection wavelength, injection volume and column temperature varied within a realistic range, and the quantitative influence of the variables was determined. The results of the robustness study are summarized in Table 5. It shows that no significant changes were observed in the chromatographic parameters of each analyte.

Table 5 Stability studies of drugs by the proposed method

Storage	40 °C and 75% humidity				30 °C and 65% humidity
	DLZ	ROS	ATR	SIM	DLZ	ROS	ATR	SIM
	% Recovery
3 months	100.21	99.07	100.11	98.6	99.55	100.08	100.41	99.38
	98.44	98.62	99.28	99.2	99.18	98.47	99.03	100.21
	99.67	99.33	98.96	100.2	98.63	99.62	99.51	99.44
	99.41	98.49	99.469	99.5	99.44	99.53	99.39	99.62
	98.97	100.24	98.74	100.4	98.94	98.69	99.28	99.51
	99.51	100.17	99.48	98.74	100.37	100.34	100.27	98.69
	99.09	99.29	99.32	99.57	99.62	99.58	9894	98.94
6 months	99.62	99.58	100.37	99.44	100.35	99.69	98.96	99.17
	100.37	98.39	98.74	98.86	99.26	99.37	99.49	99.28
	98.49	99.44	99.25	100.4	99.74	99.59	99.72	99.61
	98.49	98.85	99.14	98.66	99.27	99.22	100.48	98.79
	100.31	100.63	99.3	99.28	100.31	100.18	99.58	100.36
	100.24	99.48	100.38	99.39	99.57	99.65	99.12	99.52
	99.68	99.26	100.41	99.52	99.48	99.39	99.86	99.48

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Seven lots of commercially available drugs were retained for stability studies for up to six months duration. Two types of study were carried out, one accelerated condition and one long-term condition as described by ICH.⁵¹ Both the studies showed that all the drugs were stable under mentioned conditions, % recoveries of all the drugs obtained almost 100% (Table 5).

Serum drug analysis. The availability of all the drugs from pooled human serum was determined by the stated chromatographic conditions. Blood samples of ten healthy volunteers were collected. Volunteers (age range 22–25 years) were non-smokers, not involved in any strenuous activity and not taking any other medicines. Multiple blood samples (10 mL) were collected in evacuated glass tubes through an internal cannula placed in the forearm veins or directly from a vein. The blood was then slightly shaken and centrifuged at 3000 rpm for 10 min and the plasma separated. The obtained plasma was processed as described above and stored at −20 °C pending analysis.

Conclusion

Depicts a new RP-HPLC method with UV detection for the simultaneous determination of diltiazem and statins which is found to be linear, precise, and robust. The method was applied successfully to measure serum diltiazem and statins concentrations. A short analysis time <10 min allows application in routine and quality control analyses of finished products.

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