A novel and highly sensitive high-performance thin-layer chromatographic (HPTLC) method was developed and validated to quantify a combination of five pharmaceutical mixtures spiked to human plasma. The compounds comprised Amlodipine (AML) along with five angiotensin II receptor antagonist drugs (AIIRAs), namely Olmesartan (OLM), Telmisartan (TLM), Candesartan (CAN), Losartan (LOS), and Irbesartan (IRB). HPTLC was performed on silica gel 60 F254 plates using a mobile phase of Toluene: ethyl acetate: methanol: acetone: acetic acid (6:1.5:1:0.5:1, v/v/v/v/v). In a pioneering move, a reflectance/fluorescence detection mode was employed to identify two concurrently administered drugs at different pH levels for the first time. This method utilized the same chromatographic system, incorporating a specific measurement for AML at a neutral medium to achieve its maximum fluorescence at a 360 nm excitation wavelength, and measuring emission using a 540 nm optical filter. The process involved obtaining a very low fluorescence response from AIIRA. Subsequently, to enhance AIIRA's fluorescence, the plate was sprayed with perchloric acid to transition to a strong acidic medium, ultimately attaining the maximum fluorescence of AIIRA using various excitation wavelengths and a 400 nm emission filter. Through this strategic process, we could optimize the fluorescence signals of both drugs, thereby elevating the sensitivity of detection for this drug combination. AML demonstrated a linear range of 18-300 ng/band, while AIIRAs drugs exhibited a linear range of 6-150 ng/band. The method satisfied the International Conference on Harmonization (ICH) criteria for recovery, precision, repeatability, and robustness, showcasing exceptional sensitivity. The approach was successfully applied to quantify AML and AIIRAs drugs in both bulk drug and plasma samples, achieving high recovery percentages and minimal standard deviations.
Vilazodone is a recently approved antidepressant medicine used for treating major depressive disorder. A simple, extremely sensitive, accurate and green spectrofluorimetric method was constructed for its determination through formation of ion-pair complex with erythrosine B. The formation of ion-pair complex lowers the dye's native fluorescence emission measured at 552 nm (λ ex = 530 nm). In terms of analysis, the system's parameters for producing the vilazodone-erythrosine B complex have been optimized. The reaction was carried out in Teorell-Stenhagen buffering solution (pH 4.6). The fluorescence emission intensity of the dye decreased linearly in the range of 20 - 600 ng mL-1 and the correlation coefficient was 0.9999. The quantitation and detection limit values were 18.5 and 6.1 ng mL-1, respectively. The proposed strategy has been validated according to the ICH criteria. The proposed technique was thoroughly employed for evaluating vilazodone in raw material and pharmaceutical tablet dosage form. Furthermore, it was also successfully used for content uniformity testing. Lastly, using four advanced tools namely the Eco-Scale, the National Environmental Method Index (NEMI), the Green Analytical Procedure Index (GAPI), and the Analytical Greenness metric approach (AGREE), the greenness of the established technique was evaluated.
A green-complied spectrofluorimetric approach for quantification of the antidepressant, fluvoxamine, has been established. The method that has been suggested relies on the development of an association complex between fluvoxamine and erythrosine B in an acetate buffer solution. After being excited at 530 nm, the quenching in erythrosine B's native fluorescence caused by complex formation with fluvoxamine was detected at a wavelength of 552 nm. The values of fluorescence quenching at the most optimal reaction conditions were rectilinear at the concentration range of 0.2-2.0 µg mL-1, with a good correlation coefficient (r = 0.9998). The detection limit for the method was 0.03 µg mL-1 while the quantitation limit was 0.09 µg mL-1. The suggested approach has been validated according to the ICH. The established approach was effectively used to determine the drug under study in its dosage form with an average percent recovery of 98.92 ± 0.87 (n = 5), with no effect caused by the existing excipients. The proposed approach was also successfully used for the content uniformity test.
A spectrofluorimetric approach that is sensitive, simple, validated, and cost-effective has been proposed for the estimation of amlodipine (AML) and perindopril (PER) in their bulk powders, pharmaceutical formulations, and spiked human plasma. The recommended approach utilized the quantitative quenching effect of the two cited drugs on the fluorescence intensity of erythrosine B, as a result of complex binary reactions among each drug with erythrosine B at pH 3.5 (Teorell and Stenhagen buffer). The quenching of erythrosine B fluorescence was recorded at 554 nm after excitation at 527 nm. The calibration curve was detected in the range 0.25-3.0 µg ml-1 , with a correlation coefficient of 0.9996 for AML, and 0.1-1.5 µg ml-1 , with a correlation coefficient of 0.9996 for PER. The established spectrofluorimetric approach was validated for the estimation of the cited drugs with high sensitivity regarding International Council on Harmonization guidelines. Therefore, the established approach could be utilized for quality control of the cited drugs in their pharmaceutical formulations.
Amlodipine , Leukemia, Myeloid, Acute , Humans , Perindopril , Erythrosine , Spectrometry, Fluorescence , Pharmaceutical Preparations
In the present study, a novel spectrofluorimetric approach was designed for the analysis of captopril as a thiol-containing compound. The approach is based on the formation of a ternary fluorescent compound between the target thiol compound, ortho-phthaldehyde, and a suitable primary amine-containing compound. The produced 1-thio-alkyl-isoindole derivative exhibited very high emission activity in a faintly alkaline aqueous solution that could be monitored at 448 nm (excitation at 334 nm). 2-Amino-6-methyl-6-hydroxy-heptane was selected as the primary amine candidate that gave the high fluorescence intensity with the stability of the formed product. At the optimal experimental condition, the intensity of fluorescence of the formed product was linearly related to the concentration of captopril in 20-450 ng mL-1 range. Commercial pharmaceutical tablets were analyzed, and the obtained results agreed with those of the published method regarding precision and accuracy. The mechanism of the reaction was discussed. In addition, the greenness of the approach was rated following eco-scale criteria.
A new sensitive spectrofluorimetric technique has been established for the estimation of midodrine hydrochloride. This method depends on the condensation reaction of ortho-phthalaldehyde with the primary aliphatic amine of midodrine in the presence of 2-mercapto-ethanol. The pH of the medium was adjusted to 9.0 using 0.1 M borate buffer. The fluorescence of the product was measured at wavelength of 451 nm after excitation at 334 nm. The method was rectilinear over a concentrations range of 0.1 to 1.5 µg mL-1. The lower detection and quantitation limits were 31 and 94 ng mL-1, respectively. The method was investigated following the guidelines of the International Council of Harmonization. Analysis of commercial tablet dosage form was carried out successfully by the current method with excellent recovery percentages and with no influence from coexisted pharmaceutical additives .This method was used to evaluate the uniformity of the contents of commercial tablets according to the United States Pharmacopoeia.
Midodrine , Biological Assay , Indicators and Reagents , Midodrine/analysis , Spectrometry, Fluorescence/methods , Tablets/analysis
A novel selective and highly sensitive TLC densitometric method with reflectance/fluorescence detection was developed to separate and quantify montlukast (MONT) and loratadine (LOR). Separation of the studied drugs was performed on precoated silica gel TLC plates using chloroform-ethyl acetate (8 :2 v/v) as a mobile phase. MONT quantification was carried out by measuring emission using 400 nm optical filter after excitation at 340 nm. Enhancement of the week LOR fluorescence was performed through adequate spraying the chromatograms with 0.2 M perchloric acid leading to enhancing sensitivity by 17 folds compared to the reported HPTLC methods with absorbance detection. The scanner was set at 275 nm excitation wavelength and 400 nm optical filter. Detection of both drugs on the same plate separately at different pH conditions was utilized for the first time. Maximum fluorescence was achieved for each of them and this enhances detection sensitivity for both drugs. The linear regression analysis data of the studied drugs produced a good linear relationship with correlation coefficients of 0.996 for MONT and 0.998 for LOR over the concentration range of 6 - 150 ng/band for MONT and 15 - 120 ng/band for LOR. Limit of detection values were 1.7 and 4.5 ng /band for MONT and LOR respectively. The developed method enabled the detection of MONT and LOR in human plasma samples within linear concentrations ranged from 7 to 140 ng/ band and 16 to 50 ng/ band with detection limits of 1.9 and 4.8 ng/band for MONT and LOR respectively. The analytical performance of the proposed method was evaluated according to the International Council for Harmonization (ICH). The method was successfully applied for the analysis of the studied drugs in spiked human plasma and good recovery percentages were obtained indicating that there is no interference from plasma constituents. Therefore the method can be applied for in vivo analysis and pharmacokinetic study.
The antihypotensive drug heptaminol was determined using a spectrofluorimetric method and ortho-phthaladehyde as a fluorescence probe. The drug was mixed with the reagent in the presence of 2-mercaptoethanol and the reaction was carried out in slightly alkaline aqueous solution containing 0.1 M sodium hydroxide. The resulting product exhibited high fluorescence activity that was measured at 451 nm after excitation at 334 nm. The linearity range of the method was 5-100 ng ml-1 with a lower detection limit of 1.8 ng ml-1 . The procedure was evaluated according to the International Council of Harmonization guidelines. The proposed method was applied to analyze the drug in pharmaceutical tablets and oral drops. In addition, the present study represents the first spectrofluorimetric method for the determination of the cited drug in real human plasma. The method provided high recovery percentages without any interference from coexisting pharmaceutical excipients or the components of human plasma.
Heptaminol , Fluorescent Dyes , Humans , Plasma , Spectrometry, Fluorescence , Tablets
A natural, accurate, extremely rapid, and precise spectrofluorometric method has been developed and validated for determination of salmeterol (SAL) xinafoate in its medicinal commercial form and spiked human plasma. The native SAL fluorescence has been measured at 415 nm (after 340 nm as excitation) in distilled water. Different factors affecting the native fluorescence consistency of SAL were surveyed and optimized. The suggested procedure was capable of SAL determination over concentrations ranging 200-2000 ng.mL-1 with excellent correlation coefficient of 0.9995. The limits of detection and quantification were estimated as 44.44 ng mL-1 and 134.66 ng mL-1 , respectively. The method has good accuracy and precision. The green spectrofluorometric method was used for determination of SAL in its commercial preparations and the results were in accordance with other reported methods regarding accuracy and precision. Moreover, the proposed procedure was enforced for stability indicating assay of SAL and for SAL determination in spiked human plasma. Nearly no cost, high sensitivity, and wide application make the proposed method ideally suited for analysis of SAL in quality control laboratories.
Spectrometry, Fluorescence , Humans , Salmeterol Xinafoate
A new fluorimetric procedure is described for analysis of milnacipran in its bulk, tablet dosage forms, as well as in biological human samples such as plasma and urine. The suggested method relies on the construction of a derivative with strong fluorescence called dihydropyridine derivative. This derivative resulted from the interaction of the primary amino group in the studied drug and acetylacetone/formaldehyde in McIlvaine buffer (pH 5). The fluorescent dihydropyridine derivative was measured at 470 nm. Influences of experimental variables namely pH, reagent concentration and temperature were examined and optimized. The calibration curve showed linearity over the range of 0.15-1.25 µg ml-1 of milnacipran with an R2 value of 0.9998. The detection limit was 0.02 µg ml-1 and the determination limit was 0.07 µg ml-1 . The developed procedure was successfully used in the assay of the studied drug in Avermilan® tablets with excellent selectivity. In addition, the reaction was applied to estimate the drug in spiked human plasma and urine with mean percentage recoveries of 100.04 ± 1.61 and 99.78 ± 0.81% for urine and plasma, respectively.
Fibromyalgia , Fibromyalgia/drug therapy , Formaldehyde , Humans , Milnacipran , Spectrometry, Fluorescence , Tablets
A validated straightforward and sensitive spectrofluorimetric procedure was developed to assay trifluoperazine hydrochloride, promethazine hydrochloride, and perazine maleate. The procedure was dependent on oxidation of the investigated phenothiazines using a known excess of ammonium cerium sulfate as oxidizing agent and overseeing the fluorescence intensity of the resultant Ce3+ ion as the product of this reaction at λcx = 254 nm. and λem = 355 nm. Various parameters controlling the reaction were investigated and optimized. Linear calibration graphs were found in the general concentration range 5-30 ng/ml with a general correlation coefficient range 0.9994-0.9995. Limits of detection were 0.97, 0.70 and 0.56 ng/ml, whereas limits of quantification were 3.24, 2.12 and 1.89 ng/ml for trifluoperazine hydrochloride, promethazine hydrochloride and perazine maleate, respectively. The procedure was implemented successfully for analyses of the cited drugs in their trade dosage preparations such as tablets and syrups. The effect of possible interference from common excipients and their sulfoxide oxidized product was studied and the procedure showed good recovery of the drugs under study in their available dosage preparations. The possible effect of structure variation of the studied drugs on the experimental conditions and sensitivity observed with each one was also discussed.
Antipsychotic Agents , Phenothiazines , Spectrometry, Fluorescence , Sulfoxides , Tablets
A novel sensitive and simple spectrofluorimetric method has been developed then validated for the determination of trimetazidine in pure form and its tablets. This method is found on the reaction between trimetazidine's secondary amine moiety with NBD-Cl reagent, using borate buffer at pH 8.0 yielding a highly fluorescent product whose fluorescence intensity was measured at 526 nm (excitation at 466 nm). A calibration curve plotted showed that the linear range of the presented method was (50-700 ng/ml) with a correlation coefficient of 0.9998. The limits of detection (LOD) and limits of quantitation (LOQ) values were 15.01 and 45.50 ng/ml respectively. The presented approach was validated according to ICH guidelines and successfully applied for determining trimetazidine in its tablets with a mean percentage recovery of 99.65% ± 1.04, 99.23% ± 0.80 and 98.33% ± 1.03 for Metacardia® (20 mg), Vastor ® (20 mg) and Tricardia® (20 mg) tablets respectively. Finally, the proposed method was adopted to study the content uniformity test according to USP guidelines.
Trimetazidine , Indicators and Reagents , Limit of Detection , Reproducibility of Results , Spectrometry, Fluorescence , Tablets
A sensitive, simple, accurate and less expensive fluorimetric method was designed and validated for analysis of heptaminol HCl in both its pure and dosage forms, as well as in human plasma. The main principle used in the proposed approach was the condensation reaction between heptaminol's primary amino moiety and ethyl acetoacetate/formaldehyde reagents, giving a derivative that was highly fluorescent at 416 nm after excitation at 350 nm. Various experimental parameters that affected either the product's development or its stability were evaluated and optimized. The constructed calibration curve was linear over the range 0.2-2 µg/ml, with a good correlation coefficient (0.9996). Both the calculated limit of detection and limit of quantitation were 0.06 and 0.18 µg/ml, respectively. The presented approach was a success when used to determine Corasore® tablets and was validated according to International Council for Harmonisation guidelines.
Heptaminol , Formaldehyde , Humans , Indicators and Reagents , Spectrometry, Fluorescence , Tablets
An innovative approach to determine heptaminol spectrofluorimetrically was developed, determining the optimum conditions needed, then validated for determination of heptaminol in its pure form, its tablets and in spiked human plasma. The presented method is based on the reaction between fluorescamine reagent with the primary amine group found in heptaminol, using a borate buffer at pH 9.0 that yields a highly fluorescent product, fluorescence was measured at 471â¯nm after excitation at 393â¯nm. The linearity of the constructed calibration curve was (75-850â¯ng/ml) with LOD and LOQ values 23.85 and 72.29â¯ng/ml respectively. The method was validated following the International Council for Harmonisation (ICH) guidelines indicating good accuracy and precision. Finally, the presented approach was adapted for in vitro study of heptaminol in spiked human plasma with a mean percentage recovery 100.52⯱â¯1.19% as well as in its tablets with a mean percentage recovery 99.47⯱â¯1.25%.
Fluorescamine/chemistry , Fluorescent Dyes/chemistry , Heptaminol/blood , Spectrometry, Fluorescence/methods , Buffers , Heptaminol/chemistry , Humans , Hydrogen-Ion Concentration , Reproducibility of Results , Solvents/chemistry , Time Factors
A novel, simple, sensitive method was developed for determining midodrine spectrofluorimetrically in both its raw pure form and its tablet form. This study is based on the native fluorescence of midodrine and discusses the optimum solvent used and the wavelength range. The presented method was then validated with respect to linearity, accuracy, precision, and limits of detection and quantitation. The constructed calibration curve showed a linear range of 0.1-2.0 µg/ml. The limit of detection and limit of quantitation values were 0.03 and 0.10 µg/ml respectively. Finally, content uniformity testing was applied according to the United States Pharmacopoeia by adapting the presented method.
Midodrine/analysis , Calibration , Drug Compounding , Fluorescence , Spectrometry, Fluorescence , Tablets/analysis
A novel sensitive and simple spectrofluorimetric method was developed then validated for determination of midodrine in both its authentic pure form and its tablets. This method is based on the reaction between midodrine's aliphatic primary amine moiety with fluorescamine reagent, using borate buffer at pH 7.8 and yielding a highly fluorescent product whose fluorescence intensity was measured at 462 nm after excitation at 388 nm. This method represents the first attempt for determination of midodrine spectrofluorimetrically. A calibration curve was constructed showing that the linear range was 0.2-3.0 µg/ml. The limit of detection and limit of quantitation values were 0.06 and 0.19 µg/ml respectively. The correlation coefficient (r) and the determination coefficient (r2 ) values were 0.9992 and 0.9984 respectively. The proposed method was validated according to ICH guidelines and successfully applied for determination of midodrine in its tablets with an overall % recovery of 99.56 ± 0.95. Finally, the presented method was adapted to study the content uniformity test according to United States Pharmacopeia guidelines.
Fluorescamine/chemistry , Midodrine/analysis , Spectrometry, Fluorescence/methods , Buffers , Calibration , Hydrogen-Ion Concentration , Indicators and Reagents/chemistry , Limit of Detection , Reproducibility of Results , Sensitivity and Specificity , Solvents/chemistry , Tablets/analysis , Time Factors
Women are the most ones who susceptible to a common syndrome called fibromyalgia syndrome, up to 90% of all people with fibromyalgia are women. It affects mainly muscles and soft tissue and cause for them muscle pain, sleep problems and painful tender points. Milnacipran is acting as a serotonin-norepinephrine reuptake inhibitor (SNRI) therefore, it is recommended for the treatment of this syndrome. The widespread use of this compound in our market requires the development and validation of a simple, sensitive, cheap, fast and reproducible spectrofluorimetric method for the assay of milnacipran hydrochloride in its pure state, pharmaceutical tablets and spiked human urine and plasma. In the current work ninhydrin and phenylacetaldehyde in Teorell and Stenhagen buffer (pHâ¯7) reacts with the amino moiety of milnacipran through a sensitive condensation reaction resulting in formation of a fluorescent product, which exhibits its fluorescence emission intensity at 465â¯nm after excitation at 390â¯nm. It is observed that, in the concentration range 0.5 to 3.0⯵gmL-1, the constructed calibration curve was linear with a good correlation coefficient (0.9998). The condensation reaction was successfully applied for the assay of the studied drug in Avermilan® tablets, spiked human urine and plasma without interference from the components of the sample matrix with average percentage recoveries of 101.73⯱â¯0.56 and 100.55⯱â¯0.64 for urine and plasma, respectively.
Milnacipran/blood , Milnacipran/urine , Ninhydrin/chemistry , Spectrometry, Fluorescence/methods , Humans , Limit of Detection , Linear Models , Reproducibility of Results , Selective Serotonin Reuptake Inhibitors/blood , Selective Serotonin Reuptake Inhibitors/urine , Tablets
For the determination of heptaminol (HEP) in its authentic and dosage form as well as in human plasma, a new simple, sensitive and cheap fluorimetric method of analysis was developed and validated. The presented method is based on the reaction between aliphatic primary amino moiety present in HEP with ninhydrin and phenylacetaldehyde using Torell and Stenhagen buffer at pH 8.2 that yields a highly fluorescent derivative which after excitation at 390 nm showed a fluorescence emission at 464 nm. The effects of various experimental factors on both the development and stability of the fluorescent product was evaluated and optimized. In the concentration range (0.5-6.0 µg/ml), the constructed calibration curve was linear with a good correlation coefficient (0.9997) and the calculated limit of detection (LOD) and limit of quantitation (LOQ) were 0.14 and 0.43 respectively. The presented method was successfully applied for determination of Corasore® tablets and validated according to ICH guidelines.
Heptaminol/blood , Ninhydrin/chemistry , Heptaminol/chemistry , Humans , Molecular Structure , Spectrometry, Fluorescence
One of the most commonly used drugs in treatment of schizophrenia is flupentixol dihydrochloride, therefore it is important to develop a simple, low cost and sensitive spectrofluorimetric method for the estimation of flupentixol dihydrochloride. The yellow fluorescent product that is generated from the nucleophilic substitution reaction of the free lone pair of the alcoholic hydroxyl group of the drug and 4-chloro-7-nitrobenzofurazan (NBD-Cl) in Mcllvaine buffer pH 7.0 was estimated at 510 nm (λex 460 nm). The variables that affect the development of the reaction product were explored and optimized. The linear range of this method was 0.5-2.5 µg ml-1 with a limit of quantitation equal to 0.29 µg ml-1 . Our method was successfully applied for the assurance of flupentixol in tablet form with average percentage recovery of 99.08 ± 1.01% without obstruction from the basic excipients exhibits. Furthermore, our strategy was extended to study the content uniformity testing of flupentixol in Fluaxnol® tablets.
Benzoxazoles/chemistry , Fluorescent Dyes/chemistry , Flupenthixol/analysis , Hydrochloric Acid/analysis , Molecular Structure , Spectrometry, Fluorescence
A simple and sensitive spectrofluorimetric method has been development for the assurance of quetiapine fumarate (QTF). The proposed method was utilized for measuring the fluorescence intensity of the yellow fluorescent product at 510nm (λex 470nm). The fluorescent product has resulted from the nucleophilic substitution reaction of QTF with 4-chloro-7-nitrobenzofurazane (NBD-Cl) in Mcllvaine buffer (pH7.0). The diverse variables influencing the development of the reaction product were deliberately changed and optimized. The linear concentration range of the proposed method was of 0.2-2.0µgml-1.The limits of detection and quantitation were 0.05 and 0.17µgml-1, respectively. The proposed method was applied for the assurance of QTF in its tablets without interference from basic excipients. In addition, the proposed method was used for in vitro analysis of the QTF in spiked human plasma, the percent mean recovery was (n=3) 98.82±1.484%.
Quetiapine Fumarate/blood , Spectrometry, Fluorescence/methods , 4-Chloro-7-nitrobenzofurazan , Acetone , Humans , Hydrogen-Ion Concentration , Limit of Detection , Linear Models , Reproducibility of Results
