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Sensitive Analysis of Azelnidipine and Related Derivative in Human Plasma by Ultra-Performance Liquid Chromatography-Tandam Mass Spectrometry
Abstract
A rapid and sensitive method for analysis of azelnidipine in human plasma by ultra-performance liquid chromatography tandem mass spectrometry is presented. After pretreatment of a plasma sample by solid-phase extraction, azelnidipine was analyzed by the system with a C18 column. This method gave satisfactory recovery rates, reproducibility and good linearity of calibration curve in the range of 0.01-10 ng/mL for quality control samples spiked with azelnidipine. The detection limit was as low as 1 pg/mL. This method seems very useful in forensic and clinical toxicology and pharmacokinetic studies.
... Azelnidipine and metoprolol succinate can be determined separately or in combination with other medications using a variety of analytical techniques. Azelnidipine and metoprolol succinate cannot be determined from a synthetic mixture using any analytical approach, according to a thorough study of the literature [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. Additionally, HPLC is more practical than other chromatographic procedures since it is a process that is incredibly quick, efficient, and accurate. ...
... Suneetha G [15] et al., specifies the method for analysis of azelnidipine in human plasma by ultra-performance liquid chromatography tandem mass spectrometry. azelnidipine was analyzed by the system with a C18 column. ...
Azelnidipine (AZEL) is chemically (±)-3-(1-diphenylmethylazetidin3-yl) 5-isopropy12-amino-1, 4-dihydro-6-methyl-4-(3-nitrophenyl) 3,5-pyridinedicarboxylate. It is a dihydropyridine (DHP) type of calcium channel blocker (CCB) used for the treatment of hypertension. AZEL has two enantiomers due to an asymmetric carbon at the 4-position of the DHP ring. The pharmacological action of AZEL resides in the (R)-enantiomer. This is in marked contrast to other CCBs in which the (S)-enantiomer is responsible for the biological activity. The peculiar three-dimensional structure of the active enantiomer of AZEL may be related to its unique pharmacological features that are not shared by other DHPs such as long lasting reduction in blood pressure, decreased heart rate and antiatherosclerotic effect. AZEL also shows diuretic effect by increasing urine volume and thus reduction in retention of ions. Some analytical methods for the quantitative determination of Azelnidipine in pharmaceutical formulations like UV, LCMS/MS, RP-HPLC, HPLC-MS/MS, UFLC, LC-ESI-MS
Hypertension is the most common regulating risk factor for cardiovascular disease (CVD) and death; the increased risk associated with blood pressure (BP) elevation can be greatly reduced by treatment with antihypertensive drugs that lower both BP and related target organ damage. New Ca2+ channel antagonists have been recently developed, especially in the DHP compounds that have considerable higher vascular selectivity, slower onset and longer duration of hypotensive action. The antihypertensive effect of azelnidipine is primarily based on the inhibition of trans-membrane Ca2+ influx through the voltage-dependent channels of vascular smooth muscles. Ca2+ channels are categorized into several subtypes, including L-type, T-type, N-type, P/Q-type, and R-type Ca2+ channels depend on their electrophysiological properties. Clinical studies have demonstrated that azelnidipine markedly reduced heart rate and proteinuria in hypertensive patients by inhibiting sympathetic nerve activity. Azelnidipine has also been confirmed to have cardio-protective, neuroprotective, and anti-atherosclerotic properties, and has also been found to prevent insulin resistance.
A spectrofluorimetric method using fluorescent carbon dots (CDs) was developed for the selective detection of azelnidipine (AZEL) pharmaceutical in the presence of other drugs. In this study, N‐doped CDs (N‐CDs) were synthesized through a single‐step hydrothermal process, using citric acid and urea as precursor materials. The prepared N‐CDs showed a highly intense blue fluorescence emission at 447 nm, with a photoluminescence quantum yield of ~21.15% and a fluorescence lifetime of 0.47 ns. The N‐CDs showed selective fluorescence quenching in the presence of all three antihypertensive drugs, which was used as a successful detection platform for the analysis of AZEL. The photophysical properties, UV–vis light absorbance, fluorescence emission, and lifetime measurements support the interaction between N‐CDs and AZEL, leading to fluorescence quenching of N‐CDs as a result of ground‐state complex formation followed by a static fluorescence quenching phenomenon. The detection platform showed linearity in the range 10–200 μg/ml ( R ² = 0.9837). The developed method was effectively utilized for the quantitative analysis of AZEL in commercially available pharmaceutical tablets, yielding results that closely align with those obtained from the standard method (UV spectroscopy). With a score of 0.76 on the ‘Analytical GREEnness (AGREE)’ scale, the developed analytical method, incorporating 12 distinct green analytical chemistry components, stands out as an important technique for estimating AZEL.
An analytical method based on reverse phase-high performance liquid chromatography (RP-HPLC) has been developed and subsequently validated, which was found to be simple and rapid for the simultaneous quantification of azelnidipine and telmisartan in pharmaceutical dosage form. The separation was performed on an Intersil C18 column (250 × 4.6mm, i.d., 5µm) utilizing the mobile phase having composition 70 volumes of acetonitrile and 30 volumes of 5 millimolar phosphate buffer pH 4.6. The chromatographic analysis was carried on isocratic elution at a flow rate of 1mL/min. Detection was carried out with UV detector at 255nm, and linearity was found at concentration ranges of 10-50µg/ml for AZL and 20-100µg/ml for TEL. The recoveries obtained were 99.48‒100.22% for AZL, and 99.62 – 99.88% for TEL. No interference was found by the excipients in the formulation. The method was validated as per guidelines framed by International conference of harmonization for the parameter accuracy, precision, specificity, robustness, limits of detection and quantitation. The developed RP-HPLC method was applied in the analysis of commercial pharmaceutical products containing AZL and TEL and found to be efficient from recovery results.
High blood pressure, also called hypertension, is a common condition that is characterized by having a higher amount of pressure in blood vessels than normal. Hypertension (HT) is a very common disorder, particularly past middle age. It is not a disease in itself, but is an important risk factor for cardiovascular mortality and morbidity. For improvement activity of hypertension, Azelnidipine and Telmisartan newer combination in market, which is effective in Hypertension. This combination was developed to improve medication for Stage II Hypertension. Azelnidipine is Ca2+ channel blocker and chemically 3-[1-(Benzyldrylazetidin-3-yl] 5-isopropyl- 2- amino 6 methyl-4-(3-nitrophenyl)-1,4- dihydropyridine-3, 5dicarboxylate.Telmisartan is AT1-receptor blocker and Chemically2-{4-[[4-methyl-6-(1-methylbenzimidazol-2yl)-2–propylbenzimidazol-1yl] methyl] biphenyl)-benzoic acid. It provides information about different analytical method development like UV spectrophotometry, HPTLC, HPLC, and LC-MS methods reported for Azelnidipine and Telmisartan for individual and other drug combination. All reported methods found to be simple, accurate, economic, precise and reproducible in nature. This Review focuses on recent development in analytical method development for Azelnidipine and Telmisartan, and there were two methods reported for this combination as per our knowledge.
Backgroound
Products with multiple active substances mixed in a single dosage form are fixed-dose combinations. These are justified for a variety of reasons. These include a) increasing therapeutic efficacy, b) lowering adverse drug effects, c) pharmacokinetic advantages, d) lowering pill load, e) lowering individual drug doses, and f) lowering drug resistance development.
Objective
A recently approved fixed dose combination of azelnidipine (8 mg) and chlorthalidone (6.25 or 12.5 mg) is indicated to treat hypertension. Individual quantification methods for azelnidipine and chlorthalidone are available, but no practical and acceptable analytical approach for their combination has been documented. As a result, the goal of this literature review was to gather information on the numerous analytical instrumental approaches utilized to quantify azelnidipine and chlorthalidone in diverse matrices individually. The scientific community could use this information to design a new analytical method for analysing the recently approved combination.
Methods
Authors have explored various scientific databases to obtain information on analytical methods.
Results
The methods listed for azelnidipine and chlorthalidone are spectroscopy, high-performance liquid chromatography, hyphenated techniques, high-performance thin-layer chromatography, thin-layer chromatography, and a few other approaches. For azelnidipine and chlorthalidone, there were 26 and 46 research papers reported, respectively.
A novel simple, rapid, sensitive and stability-indicating RP-HPLC method was developed and validated for the determination of azelnidipine (ALDP) and its impurities 1 and 2. Resolution of drug, its potential impurities and degradation products were achieved by RP-HPLC on was performed on Prontosil ODS C18 column (250 mm x 4.6 mm, 5µ) using a mobile phase consisting of methanol and 0.1M sodium acetate 40: 60 (v/v) at a flow rate of 1 ml/min and 231 nm of UV detector. Validation of the method was performed along with formulation analysis and forced degradation studies. The calibration curves of ALDP were linear over a concentration range of 50-300 µg/mL. The method was rapid with a retention time of the impurity 2, impurity and ALDP observed at 3.60, 5.15 and 6.90 min, respectively. The method was applied for the impurities determination in drug tablets and for degradation products determination in a stability study of ALDP. The impurity content in the tablets was quantified as 0.1% of total drug. The method can also be used for rapid and accurate quantification of ALDP in its tablets during stability testing.
The literature survey explains that there is not any stability indicating method reportedly for combination of Azelnidipine and Telmisartan till date. Validation and development of stability indicating analytical methods is possible as per ICH Guidelines. There are several of Spectroscopic methods such as Ultraviolet Spectroscopy, Mass spectroscopy, infrared spectroscopy, Nuclear magnetic resonance spectroscopy and Chromatographic methods such as High performance liquid chromatography, Thin layer Chromatography, High Performance thin layer chromatography, Gas chromatography and Ultra performance liquid chromatography etc. used for stability indicating method development and validation.
CS-905 is a potent dihydropyridine calcium blocker that has a gradual and long-lasting antihypertensive action with little tachycardia in SHR. In this study, we investigated chronic and acute effects of CS-905 on renal functions in SHR. To examine the chronic effects, 23 week-old male SHR were treated with CS-905 (1 or 3 mg/kg/day, p.o.) or 0.3% CMC (carboxymethylcellulose). After the 15 weektreatment, the agent dose-relatedly lowered systolic blood pressure measured 24 hr after the final administration (184±2 and 173±3 mmHg at 1 and 3 mg/kg/day vs. 218±4 mmHg for the control group). Natriuresis and the reduction of urinary protein excretion were also observed in the CS-905 treated groups. Urinary NAG (N-acetyl-β-D-glucosaminidase) activity tended to decrease, but not significantly. Histopathological changes observed in the SHR kidney were reduced by chronic treatment with CS-905. On a single oral administration in 38 week-old SHR, CS-905 caused natriuresis at a dose of 3 mg/kg, but did not affect urinary protein excretion and urinary NAG activity. These effects of CS-905 on renal functions may be beneficial in the treatment of hypertension.
A sensitive gas chromatographic-mass spectrometric method for the quantitation of (+/-)-methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (OPC-13340, I), a new dihydropyridine calcium antagonist with a potent and long-acting antihypertensive and antianginal effect, was developed in order to elucidate its pharmacokinetics. Dihydropyridine calcium antagonist have been usually quantifed by this technique in the negative-ion chemical ionization mode. However, direct application of this method to quantify trace amounts of I in biological fluids completely failed, owing to its adsorption on the column and oxidation of its dihydropyridine ring. Human plasma containing I and (+/-)-[2H3]methyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (II), the internal standard, was extracted with n-hexane-diethyl ether under weakly basic conditions (pH 8). In order to prevent adsorption of the compounds on the column, (+/-)-[2H5]ethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate (III), an analogue of I, was added to the extracts as a carrier. In addition, this carrier was also effective in preventing the oxidation of I. The quantitation limit of I in human plasma by this method was found to be less than 30 pg/ml. Thus, the method is sufficiently sensitive to study the pharmacokinetics of I in humans.
CS-905 is a novel dihydropyridine calcium blocker. A single oral administration of CS-905 or nicardipine at doses of 0.3-3.0 mg/kg produced a dose-dependent reduction of blood pressure in conscious SHR. CS-905, when administered orally in conscious SHR, was more than 3 times as potent as nicardipine. Unlike the hypotensive effect of nicardipine, that of CS-905 has a gradual onset and is long-lasting, with little increase in heart rate. An intravenous administration of CS-905 also produced a hypotension with a slow onset and long duration in SHR, but CS-905 was 3 times less potent than nicardipine by intravenous administration. This difference may be attributed to the first pass effect, which was associated with nicardipine but not with CS-905. The blood pressure lowering effects of CS-905 was most potent in DOCA-salt hypertensive rats, followed by SHR, RHR and normotensive rats, in this order. CS-905 is expected to be an antihypertensive agent that is effective on a once a day regimen in clinical settings.
A sensitive, efficient, linear and reproducible capillary gas chromatographic method with electron-capture detection was developed for the quantitation of nifedipine and its primary metabolite M-I in plasma together with the urinary and principal metabolites M-II and M-III. On-column, rather than split-splitless, injection was employed to obviate oxidative degradation of nifedipine to M-I. The photosensitivity of nifedipine was re-examined under laboratory conditions and nifedipine was found to have a half-life in excess of two days when amber glassware and darkroom manipulations under red light were used. The method can determine nifedipine and its metabolites in plasma and urine after a single oral dose of 5 mg and can be applied to measure M-I production by human liver microsomes.
A highly sensitive and specific method for the determination of nilvadipine, a new dihydropyridine calcium antagonist, in human plasma is described. A deuterated analogue of nilvadipine is added to the plasma as an internal standard. The agent and its internal standard are extracted at pH 9 from the plasma into a benzene--n-hexane (1:1) mixture. The extract is analysed by fused-silica capillary column gas chromatography--negative-ion chemical-ionization mass spectrometry with methane as the reagent gas. The mass spectrometer is set to monitor the negative molecular ions of the agent and internal standard. Quantitation is possible down to 0.01 ng/ml using 1 ml of plasma. The coefficients of variation of the method are 6.4 and 2.1% at the 0.01 and 0.1 ng/ml levels, respectively. Plasma levels obtained with this method are given for four healthy volunteers who had received a 6-mg oral dose of nilvadipine.
A rapid and specific method in which reverse-phase high-performance liquid chromatography (HPLC) with UV detection was used for the simultaneous determination of nicardipine and its pyridine metabolite II in human plasma is described. Nicardipine, its pyridine metabolite II, and the internal standard were extracted from plasma and partially purified by acid-base partitioning. Final purification and quantitation were achieved by HPLC by using a reverse-phase column and a UV detector (254 nm). The extraction efficiencies for nicardipine and its pyridine metabolite II from 1 mL of plasma were 77.4 and 81.1%, respectively. The sensitivity of the assay was 5 ng/mL for both nicardipine and its pyridine metabolite II, and the linear concentration range of the assay was 5-150 ng/mL for both compounds. The low coefficients of variation (less than or equal to 5%) for samples spiked with nicardipine and its pyridine metabolite II in this concentration range demonstrate good reliability and reproducibility of the assay. The HPLC procedure has been validated by comparison with a GC-electron-capture detection (ECD) procedure, which gives the combined concentration of nicardipine-its pyridine metabolite II (total) and with an HPLC/GC-ECD procedure, which gives the concentration of its pyridine metabolite II. All three methods, which were developed in our laboratory, were used to analyze nicardipine and its pyridine metabolite II in specimens of plasma from subjects treated with nicardipine hydrochloride. Good correlations were found for concentrations of nicardipine, its pyridine metabolite II, and nicardipine plus the metabolite determined by these three procedures. The HPLC procedure is suitable for use in pharmacokinetic studies following administration of nicardipine hydrochloride to humans.
A sensitive and specific LC-MS-MS assay has been developed and validated for barnidipine (1-benzyl-3-pyrrolidinyl)methyl-2,6-dimethyl-4(m-nitrophenyl)-1,4-dihydr opyridine-3,5-dicarboxylate). The assay involves a simple and rapid solid-phase extraction procedure. Sample analysis was on a Spherisorb S3ODS2 100 mmX2 mm I.D. column, with a Finnigan TSQ 7000 mass spectrometer, using an electrospray interface and selective reaction monitoring (SRM). The intra- and inter-day precision and accuracy, determined as the coefficient of variation and relative error, respectively, were 11.8% or less. The limit of quantitation was 0.03 ng/ml, and the calibration was linear between 0.03 and 3.0 ng/ml. The method has been used successfully for the measurement of over two thousand human plasma samples from pharmacokinetic clinical trials.
An analytical methodology for nifedipine quantitation in plasma by on-line solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) is described. The SPE cartridges contain C2 and the analytes nifedipine and nitrendipine (internal standard) are separated on a C18 column with a mobile phase consisting of acetonitrile-13 mM phosphate buffer pH 7 (65:35, v/v) followed by UV detection at 338 nm. Validation of the method demonstrated good recoveries (>90%), sensitivity (limit of quantification, 2 ng/ml), based on a 500 microl sample volume, accuracy and precision (<5.5% in concentrations greater than the limit of quantitation). This methodology has been used for bioequivalence studies.
We objected: 1) To compare the effects of azelnidipine and amlodipine on 24-h blood pressure; 2) To monitor the plasma concentration vs. the time profile in order to assess the association between pharmacokinetics and hypotensive activity after administration of either drug for 6 weeks. Blood pressure and pulse rate were measured by 24-h monitoring with a portable automatic monitor in a randomized double-blind study of 46 patients with essential hypertension. Azelnidipine 16 mg (23 patients) or amlodipine 5 mg (23 patients) was administered once daily for 6 weeks. Pharmacokinetics were analyzed after the last dose was taken. Both drugs showed similar effects on the office blood pressure and pulse rate. During 24-h monitoring, both drugs caused a decrease in systolic blood pressure of 13 mmHg and had a similar hypotensive profile during the daytime period (07:00-21:30). The pulse rate decreased by 2 beats/min in the azelnidipine group, whereas it significantly increased by 4 beats/min in the amlodipine group. Similar trends in the blood pressure and pulse rate were observed during the nighttime (22:00-6:30) and over 24 h. Excessive blood pressure reduction during the nighttime was not seen in either group. The pharmacokinetic results indicated that the plasma half-life (t1/2) of amlodipine was 38.5 +/- 19.8 h and that of azelnidipine was 8.68 +/- 1.33 h. Despite this difference in pharmacokinetics, the hypotensive effects of amlodipine and azelnidipine were similar throughout the 24-h administration period.
Nifedipine, a dihydropyridine calcium channel antagonist, is widely used in the treatment of hypertension and other cardiovascular disorders. A simple, rapid, sensitive, precise and accurate HPLC method, using solid-phase extraction, for the quantitation of nifedipine in human plasma was developed and validated. The calibration graphs were linear in the 5-400 ng/ml concentration range (r>0.999). Recovery for nifedipine was greater than 93.9% and for internal standard nitrendipine was 96.1%. Intra-day and inter-day precision ranged from 1.4 to 4.2 and 3.9 to 5.6%, respectively. Intra-day and inter-day accuracy was ranged from 94.5 to 98.0 and 93.1 to 98.0%, respectively. The method was not interfered with by other plasma components and was applied for the determination of nifedipine in pharmacokinetic study after single oral administration of 10 mg nifedipine to 18 healthy male subjects.
A rapid, sensitive, robust and specific method was developed for the determination and quantitation of felodipine, in human blood plasma by liquid chromatography coupled with tandem mass spectrometry using nimodipine as internal standard. Felodipine was extracted from 0.5 mL human plasma by use of a liquid/liquid procedure using diethyl ether/hexane (80/20, v/v) as eluent. The method included a chromatographic run of 5 min using a C(18) analytical column (100 mm x 4.6 mm i.d.) and the calibration curve was linear over the range from 0.02 to 10 ng mL(-1) (r(2) > 0.994). The between-run precision, determined as relative standard deviation of replicate quality controls, was 5.7% (0.06 ng mL(-1)), 7.1% (0.6 ng mL(-1)) and 6.8% (7.5 ng mL(-1)). The between-run accuracy was +/- 0.0, 2.1 and 3.1% for the above-mentioned concentrations, respectively.
Overproduction of nitric oxide by inducible nitric oxide synthase contributes to the progression of cardiovascular disease. We investigated the effects of azelnidipine and other Ca2+-channel blockers on nitric oxide production by cultured aortic smooth muscle cells isolated from Wistar rats and human umbilical vein endothelial cells (HUVECs), using the Griess reaction and oxyhemoglobin method. Release of lactic dehydrogenase (LDH) was measured to evaluate cell damage, and immunohistochemistry was performed to examine the expression of inducible nitric oxide synthase and nitrotyrosine protein. Azelnidipine and other Ca2+-channel blockers inhibited the release of nitric oxide induced by lipopolysaccharide plus interferon-gamma. Azelnidipine inhibited it most potently among the Ca2+-channel blockers tested (azelnidipine, amlodipine, nifedipine, diltiazem, verapamil, and nicardipine) at a concentration of 10 microM. Longer stimulation with these agents induced the expression of inducible nitric oxide synthase and nitrotyrosine, with an increase of lactic dehydrogenase release, whereas azelnidipine suppressed these changes. In human umbilical vein endothelial cells, azelnidipine enhanced basal nitric oxide production by endothelial nitric oxide synthase. In conclusion, azelnidipine potently inhibited the induction of inducible nitric oxide synthase and then nitric oxide production in vascular smooth muscle cells, while enhancing constitutive nitric oxide production by endothelial cells. Azelnidipine may inhibit nitrotyrosine expression and cell damage caused by overproduction of nitric oxide, suggesting a mechanism for its cardiovascular protective effect.