Sensitive spectrofluorometric method for the determination of ascorbic acid in pharmaceutical nutritional supplements using acriflavine as a fluorescence reagent

An easily performed, specific, sensitive, rapid, reliable and inexpensive procedure for the spectrofluorometric quantitation of ascorbic acid was proposed using acriflavine as a fluorescence quenching reagent. The procedure was based on the determined quenching effect of ascorbic acid on the natural fluorescence signal of acriflavine and the reaction between ascorbic acid and acriflavine in Britton–Robinson buffer solution (pH 6) to produce an ion‐associated complex. The reduction in acriflavine fluorescence intensity was detected at 505 nm, while excitation occurred at 265 nm. The relationship between quenching fluorescence intensity (?F) and concentration of ascorbic acid was linear (R² = 0.9967) within the range 2–10 μg/ml and with a detection limit of 0.08 μg/ml. No significant interference was detected from other materials often found in pharmaceutical nutritional tablets. The obtained results were compared with those from high‐performance liquid chromatography and appeared in good agreement, with no important differences in precision or accuracy. The proposed spectrofluorimetric method was used to determine the amount of ascorbic acid in a number of commercial pharmaceutical nutritional supplement tablets with a 95% confidence performance.     

Highly sensitive HPLC method for the determination of galantamine in human plasma and urine through derivatization with dansyl chloride using fluorescence detector

A new method based on fluorescence derivatization with 5‐(dimethylamino) naphthalene‐1‐sulfonyl chloride (dansyl chloride) was developed for the quantitative determination of galantamine in human plasma and urine using high‐performance liquid chromatography. The reaction between galantamine and dansyl chloride was optimally realized in 30 min at room temperature and pH 10.5, with a reagent to galantamine molar ratio of 2.13. The derivative was extracted with dichloromethane, and the extract was dried under a nitrogen stream and dissolved in the mobile phase. Chromatographic analysis was performed with an Inertsil C₁₈ column and a mobile phase comprising 40% acetonitrile and 60% 10 mM o‐phosphoric acid, 1.2 ml/min. The injection volume was 20 μl. The derivatives were detected with a fluorescence detector (excitation 375 nm/emission 537 nm). The retention time for the dansyl derivative of galantamine was 16.8 min. Linearity was observed between 125 and 2000 ng/ml in water, urine and plasma. The limit of detection and limit of quantification for the developed method were 6.27–70.99 and 18.81–212.97 ng/ml, respectively. Per cent recovery was calculated as 95.15 for urine and 95.78 for plasma. Interday repeatability values for urine and plasma samples (n = 6) at three different concentrations were calculated as a per cent relative standard deviation of 0.24–0.59 and 0.35–0.56. The corresponding per cent relative standard deviation values for intraday repeatability were 0.13–0.51 and 0.04–0.15, respectively.     

Enantioselective analysis of ibuprofen enantiomers in mice plasma and tissues by high‐performance liquid chromatography with fluorescence detection: Application to a pharmacokinetic study

A direct fluorometric high‐performance liquid chromatography (HPLC) method was developed and validated for the analysis of ibuprofen enantiomers in mouse plasma (100 μl) and tissues (brain, liver, kidneys) using liquid–liquid extraction and 4‐tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers was accomplished in a Chiracel OJ‐H chiral column based on cellulose tris(4‐methylbenzoate) coated on 5 μm silica‐gel, 250 x 4.6 mm at 22 °C with a mobile phase composed of n‐hexane, 2‐propanol, and trifluoroacetic acid that were delivered in gradient elution at a flow rate of 1 ml min⁻¹. A fluorometric detector was set at: λ_excit. = 220 nm and λ_emis. = 290 nm. Method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within‐run and between‐run precision and accuracy. The LLOQ for the two enantiomers was 0.125 μg ml⁻¹ in plasma, 0.09 μg g⁻¹ in brain, and 0.25 μg g⁻¹ in for liver and kidney homogenates. The calibration curves showed good linearity in the ranges of each enantiomers: from 0.125 to 35 μg ml⁻¹ for plasma, 0.09–1.44 μg g⁻¹ for brain, and 0.25–20 μg g⁻¹ for liver and kidney homogenates. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice treated i.v. with 10 mg kg⁻¹ of racemate.     

Utility of a xanthene-based dye for determination of nilotinib using two spectroscopic approaches. Applications to bulk powder,capsules, and spiked human plasma

Novel, selective, facile, and precise spectroscopic approaches were validated to determine nilotinib hydrochloride, a tyrosine kinase inhibitor used to treat patients with chronic myeloid leukemia. These approaches depend on the reaction of the tertiary amine group of nilotinib with erythrosine B in the Britton–Robinson buffer at pH 4. Method I, depends on measuring the absorbance of the formed complex at 551 nm. The absorbance concentration plot showed linearity over the concentration range of 1.0 to 9.0 μg/ml. Method II, involved the measurement of the quenching of the native fluorescence of erythrosine B by adding nilotinib in an acidic medium. The fluorescence quenching of erythrosine B was measured at 549 nm after excitation at 528 nm. This approach showed excellent linearity in the concentration range of 0.04 to 0.7 μg/ml. The limit of detection values for Method I and Method II were 0.225 and 0.008 μg/ml, respectively, while the limit of quantitation values for Method I and Method II were 0.68 and 0.026 μg/ml, respectively. To get the optimal conditions, factors that may affect the formation of the ion-pairing complex were thoroughly examined. The two approaches were carefully validated following the International Conference of Harmonization (ICH Q2R1) guidelines. Statistical assessment of the results achieved using the suggested and previously published comparison approaches showed no significant difference. The approaches were successful in determining nilotinib in a pharmaceutical dosage form as well as spiked human plasma samples. The eco-friendly properties of the methods were evaluated by three different tools.     

Development of a micelle‐enhanced high‐throughput fluorometric method for determination of niclosamide using a microplate reader

The present paper describes the development and validation of a simple and sensitive micelle‐enhanced high‐throughput fluorometric method for the determination of niclosamide (NIC) in 96‐microwell plates. The proposed method is based on the reduction of the nitro group of niclosamide to an amino group using Zn/HCl to give a highly fluorescent derivative that was developed simultaneously and measured at λ_em 444 nm after excitation at λ_ex 275 nm. Tween‐80 and carboxymethylcellulose (CMC) have been used as fluorescence enhancers and greatly enhanced the fluorescence by factors of 100–150%. The different experimental conditions affecting the fluorescence reaction were carefully investigated and optimized. The proposed method showed good linearity (r²≥ 0.9997) over the concentration ranges of 1–5 and 0.5–5 μg/ml with lower detection limits of 0.01 and 0.008 μg/ml and lower quantification limits of 0.04 and 0.03 μg/ml on using Tween‐80 and or CMC, respectively. The developed high‐throughput method was successfully applied for the determination of niclosamide in both tablets and spiked plasma. The capability of the method for measuring microvolume samples made it convenient for handling a very large number of samples simultaneously. In addition, it is considered an environmentally friendly method with lower consumption of chemicals and solvents.     

Spectrofluorimetric determination of eptifibatide in human plasma and dosage form

A spectrofluorimetric method for the determination of eptifibatide is presented based on its native fluorescence. The type of solvent and the wavelength of maximum excitation and emission were carefully selected to optimize the experimental conditions. Under the specified experimental conditions, the linearities obtained between the emission intensity and the corresponding concentrations of eptifibatide were in the range 0.1–2.5 μg/ml for the calibration curve constructed for direct determination of eptifibatide in dosage form and 0.05–2.2 μg/ml for the calibration curve constructed in spiked human plasma with a good correlation coefficient (r > 0.99). The lower limit of quantification for the calibration curve constructed in human plasma was 0.05 μg/ml. Recovery results for eptifibatide in spiked plasma samples and in dosage form, represented as mean ± % RSD, were 95.17 ± 1.94 and 100.29 ± 1.33 respectively. The suggested procedures were validated according to the International Conference on Harmonization (ICH) guidelines for the direct determination of eptifibatide in its pure form and dosage form and United States Food and Drug Administration (US FDA) Guidance for Industry, Bioanalytical Method Validation for the assay of eptifibatide in human plasma.     

Simultaneous HPLC–UV determination of rhein and aceclofenac in human plasma

Diacerein and aceclofenac are prescribed for reducing the symptoms associated with osteoarthritis. We present a simple HPLC method with UV detection for simultaneous determination of rhein (the immediate metabolite of diacerein) and aceclofenac from human plasma samples. Sample preparation was accomplished through liquid–liquid extraction with ethyl acetate and chromatographic separation was performed on a reversed-phase ODS column. Mobile phase consisted of a mixture of acetate buffer and acetonitrile run under gradient at flow rate of 1.0 ml/min. Wavelength was set at 258 nm. The method was validated for linearity, accuracy, precision and stability. The calibration was linear over the range of 0.1–7.0 μg/ml for rhein and 0.5–20 μg/ml for aceclofenac using 500 μl plasma samples. Extraction recoveries were 85% for rhein and 70% for aceclofenac. The method can easily be adopted for high-throughput clinical and pharmacokinetic studies of above two-drug fixed dose combination formulations.     

Eco-friendly approach for the determination of moxifloxacin in pharmaceutical formulations and biological fluids based on fluorescence quenching of l-tryptophan

A rapid, novel and cost-effective spectrofluorimetric method developed to determine moxifloxacin (MFX) in pharmaceutical preparations because MFX in a pH 10 medium could reduce the fluorescence intensity of l -tryptophan. The maximum fluorescence excitation and emission wavelengths were found to be 280 and 363 nm respectively. A range of factors affecting fluorescence quenching and the effect of co-existing substances were investigated. Fluorescence quenching values (ΔF = F_L-tryptophan − F_{Moxi-L-tryptophan}) displayed a strong linear relationship with the MFX concentration ranging from 0.2 to 8.0 μg/ml under optimum conditions. The limit of detection was found to be 6.1 × 10⁻⁴ μg/ml. The proposed method was shown to be suitable for MFX determination in pharmaceutical tablets and biological fluids by the linearity, recovery and limit of detection. The spectrofluorimetric approach that has been developed is extremely eco-friendly, as evidenced by the fact that all the experimental components and solvents were safe for the environment.     

The convenient use of fluorescamine for spectrofluorimetric determination of midodrine hydrochloride in pure form and its tablets formulation: Application to content uniformity testing

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.     

Simple and rapid liquid chromatography method for determination of moxifloxacin in plasma

A high performance liquid chromatographic method for determination of moxifloxacin in human plasma was developed. The method involved deproteinisation of the sample with perchloric acid and analysis of the supernatant using a reversed-phase C₁₈ column (150 mm) and fluorescence detection at an excitation wavelength of 290 nm and an emission wavelength of 460 nm. The assay was specific for moxifloxacin and linear from 0.125 to 10.0 μg/ml. The relative standard deviation of intra- and inter-day assays was lower than 10%. The average recovery of moxifloxacin from plasma was 101%. Due to its simplicity, the assay can be used for pharmacokinetic studies of moxifloxacin.     

Highly sensitive spectrofluorimetric method for rapid determination of orciprenaline in biological fluids and pharmaceuticals

Orciprenaline sulphate (ORP) is a direct‐acting sympathomimetic with mainly beta‐adrenoceptor stimulant activity. It is used as a bronchodilator in the management of reversible airway obstruction. For the first time, a rapid highly sensitive spectrofluorimetric method is described that is relied on measuring the fluorescence spectra of ORP at acidic pH and without addition of any chemical reagents. The relative fluorescence intensity was measured at 310 nm and after excitation at 224 nm. ORP native fluorescence was calibrated in both water and acetonitrile as diluting solvents. The method was designed to estimate the drug in miscellaneous matrices with high accuracy and precision. Linear ranges of calibration curves were 30.0–400.0 ng/ml and 10.0–240.0 ng/ml in water and acetonitrile, respectively. The detection limits were calculated and reached as low as 3.3 and 3.1 ng/ml, respectively, representing the ultra‐sensitivity of the proposed method. This result permitted application of this method for spiked human plasma and urine and was used as a preliminary investigation with good percentage recovery (89.4–106.8%). The application was further extended to analyse ORP in its pharmaceutical formulations. The method was validated in compliance with International Council of Harmonization (ICH) Guidelines.     

Spectrofluorimetric determination of acotiamide hydrochloride trihydrate in the presence of its oxidative degradation product

Acotiamide hydrochloride trihydrate is a novel gastroprokinetic drug which has been recently approved for the treatment of patients with functional dyspepsia. This study presents the first reported to investigate the fluorimetric behavior of acotiamide hydrochloride trihydrate in the presence of its oxidative degradation product. All variables that affect fluorescence intensity were studied and optimized. The described method involved the measurement of native fluorescence of the drug in ethanol at 404 nm after excitation at 326 nm. Calibration plot was found to be linear over the concentration range 0.1–0.9 μg/ml. The specificity of the method has been tested via selective determination of the studied drug in its synthetic mixtures with its degradation product. The proposed method has been successfully applied to the analysis of the drug in its new pharmaceutical dosage form and the results have been statistically compared with the reported HPLC method showing no significant differences by applying t‐test and F‐test.     

Spectrofluorimetric method for the determination of sulpiride in pharmaceutical preparations and human plasma through derivatization with 2‐cyanoacetamide

A sensitive and accurate spectrofluorimetric method has been developed for the determination of sulpiride in pharmaceutical preparations and human plasma. The developed method is based on the derivatization reaction of 2‐cyanoacetamide with sulpiride in 30% ammonical solution. The fluorescent derivatized reaction product exhibited maximum fluorescence intensity at 379 nm after excitation at 330 nm. The optimum conditions for derivatization reactions were studied and the fluorescence intensity versus concentration plot was found to be linear over the concentration range 0.2–20.0 µg/mL with a correlation coefficient of 0.9985. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.82 and 2.73 ng/mL, respectively. The proposed method was validated according to ICH guidelines. The effects of common excipients and co‐administered drugs were also studied. The accuracy of the method was checked using the standard addition method and percent recoveries were found to be in the range of 99.00–101.25% for pharmaceutical preparations and 97.00–97.80% for spiked human plasma. The method was successfully applied to commercial formulations and the results obtained for the proposed method were compared with a high‐performance liquid chromatography reference method and statistically evaluated using the Student's t‐test for accuracy and the variance ratio F‐test for precision. A reaction pathway was also proposed. Copyright © 2012 John Wiley & Sons, Ltd.     

Utility of ninhydrin reagent for spectrofluorimetric determination of heptaminol in human plasma

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.     

Validated spectrofluorimetric and resonance Rayleigh scattering methods for determining naftidrofuryl in varied pharmaceutical samples based on its interaction with erythrosin B

Naftidrofuryl is a vasodilator medication used for treating cerebral and peripheral vascular diseases. In this study, two spectroscopical techniques, spectrofluorimetric and resonance Rayleigh scattering (RRS), were utilized to quantify naftidrofuryl in its pharmaceutical samples. The developed methodologies in this study rely on a facile process of forming an association complex between erythrosine B reagent and naftidrofuryl under acidic conditions. The fluorimetric assay is based on the ability of naftidrofuryl to quench and decrease the native fluorescence intensity of the reagent when measured at ${\lambda }_{\mathit{\text{emis}}.}$ = 550 nm ( ${\lambda }_{\mathit{\text{excit}}.}$ = 526 nm). Under similar reaction conditions, the RRS method relies on the observed amplification in the RRS spectrum of the reagent at a wavelength of 577 nm following its interaction with naftidrofuryl. The methods exhibited linearity within the ranges 0.2–1.6 μg/ml (r² = 0.999) and 0.1–1.4 μg/ml (r² = 0.9994), with limit of quantitation values of 0.146 and 0.099 μg/ml, and limit of detection values of 0.048 and 0.032 μg/ml, for the fluorometric and the RRS methods, respectively. Moreover, the quenching between the dye and naftidrofuryl was studied using Stern–Volmer analysis, and the methodologies were experimentally optimized and validated. Additionally, acceptable recoveries were achieved when the procedures were applied to determine naftidrofuryl in pharmaceutical samples.     

Determination of lipoic acid in human plasma by HPLC-ECD using liquid–liquid and solid-phase extraction: Method development,validation and optimization of experimental parameters

A rapid, inexpensive, sensitive and specific HPLC-ECD method for the determination of lipoic acid in human plasma was developed and validated over the linearity range of 0.001–10 μg/ml using naproxen sodium as an internal standard (IS). Extraction of lipoic acid and IS from plasma (250 μl) was carried out with a simple one step liquid–liquid extraction using dichloromethane. Similarly solid-phase extraction was carried out using dichloromethane as extraction solvent. The separated organic layer was dried under the stream of nitrogen at 40 °C and the residue was reconstituted with the mobile phase. Complete separation of both lipoic acid and IS at 30 °C on Discovery HS C18 RP column (250 mm × 4.6 mm, 5 μm) was achieved in 6 min using 0.05 M phosphate buffer (pH 2.5 adjusted with phosphoric acid):acetonitrile (50:50, v/v) as a mobile phase pumped at the rate of 1.5 ml/min using electrochemical detector in DC mode at the detector potential of 1.0 V. The limit of detection and limit of quantification of lipoic acid were 200 pg/ml and 1 ng/ml, respectively. While on column limit of detection and limit of quantification of lipoic acid were 10 and 50 pg/ml, respectively. The absolute recoveries of lipoic acid with liquid–liquid and solid-phase extraction were 98.43, 95.65, 101.45, and 97.36, 102.73, 100.17% at 0.5, 1 and 5 μg/ml levels, respectively. Coefficient of variations for both intra-day and inter-day were between 0.28 and 4.97%. The method is validated and will be quite suitable for the analysis of lipoic acid in the plasma of human volunteers as well as patients with diabetes and cardiovascular diseases.     

Application of π acceptors to the spectrophotometric and spectrofluorimetric determination of vincamine and naftidrofuryl oxalate in their pharmaceutical preparations

Three different spectrophotometric and two spectrofluorimetric methods have been developed and validated for the determination of vincamine (VN) and naftidrofuryl oxalate (NF) in tablets. The spectrophotometric methods depend on charge transfer complex formation between each of VN and NF with 7,7,8,8‐tetracyano‐quinodimethane (TCNQ), 2,6‐dichloroquinone‐4‐chloroimide (DCQ) and 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) at 843, 580 and 588 nm, respectively. The spectrofluorimetric methods are based on the formation of charge transfer complex between each of the two drugs and TCNQ, with measurement of the fluorophore formed at 312/375 and 284/612 nm, respectively, or with DDQ at 400/475 and 284/396 nm, respectively. In the spectrophotometric measurements, Beer's law was obeyed at concentration ranges of 1.5–16, 10–180 and 12–140 μg/ml for VN with TCNQ, DCQ, and DDQ, respectively. For NF, the corresponding concentrations were 2–28, 5–75 and 25–150 μg/ml with TCNQ, DCQ, and DDQ, respectively. In the spectrofluorimetric measurements, the ranges for VN were 0.05–0.9 and 0.3–4 μg/ml with TCNQ and DDQ, respectively, whereas for NF the ranges were 0.05–0.85 and 0.5–8 μg/ml with TCNQ and DDQ, respectively. The different experimental parameters affecting the development and stability of the formed color or fluorophore were studied and optimized and the molar ratios of the complexes were calculated. The proposed methods were validated according to ICH guidelines and were successfully applied for the determination of VN and NF in their tablet dosage forms.     

Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography

Taxifolin has been widely used in the treatment of cerebral infarction and sequelae, cerebral thrombus, coronary heart disease and angina pectoris. A reliable sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection for the pharmacokinetic study of taxifolin in rabbit plasma after enzymatic hydrolysis was developed and validated for the first time. Taxifolin, with biochanin A as the internal standard, was extracted from plasma samples by liquid/liquid extraction after hydrolysis with β-glucuronidase and sulfatase. Chromatographic separation was conducted on a Luna C18 column (4.6 mm×150 mm, 5 μm particle size) and pre-column (2.0 mm, the same sorbent). Two-step linear gradient elution with acetonitrile and 0.03% water solution of trifluoroacetic acid as mobile phase at a flow rate of 1.0 ml/min was used. The UV detector is set at 290 nm. The elution time for taxifolin and biochanin A was approximately 7.9 and 18.3 min, respectively. The calibration curve of taxifolin was linear (r>0.9997) over the range of 0.03–5.0 μg/ml in rabbit plasma. The limit of detection (LOD) and limit of quantification (LOQ) for taxifolin were 0.03 and 0.11 μg/ml, respectively. The present method was successfully applied for the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration of lipid solution to rabbits. The absolute bioavailability of taxifolin after oral administration of lipid solution was 36%.     

Simultaneous rapid high-performance liquid chromatographic determination of phenytoin and its prodrug, fosphenytoin in human plasma and ultrafiltrate

A reversed-phase high-performance liquid chromatographic assay for the simultaneous determination of phenytoin and fosphenytoin, a prodrug for phenytoin, in human plasma and plasma ultrafiltrate is described. For plasma, the method involves simple extraction of drugs with diethyl ether and evaporation of solvent, followed by injection of the reconstituted sample onto a reversed-phase C₁₈ column. Plasma ultrafiltrate is injected directly into the HPLC column. Compounds are eluted using an ion-pair mobile phase containing 20% acetonitrile. The eluent is monitored by UV absorbance at 210 nm. The fosphenytoin standard curves are linear in the concentration range 0.4 to 400 μg/ml for plasma and 0.03 to 80 μg/ml for ultrafiltrate. Phenytoin standard curves are linear from 0.08 to 40 μg/ml for plasma and from 0.02 to 5.0 μg/ml for ultrafiltrate. No interferences with the assay procedure were found in drug-free blank plasma or plasma ultrafiltrate. Relative standard deviation for replicate plasma or ultrafiltrate samples was less than 5% at concentrations above the limit of quantitation for both within- and between-run calculations.     

Micelle‐enhanced direct spectrofluorimetric method for the determination of linifanib: Application to stability studies

A new simple stability‐indicating spectrofluorimetric method has been developed and validated for the determination of the tyrosine kinase inhibitor, linifanib (LNF). The proposed method makes use of the native fluorescence characteristics of LNF in a micellar system. Compared with aqueous solutions, the fluorescence intensity of LNF was greatly enhanced upon the addition of Tween‐80. The relative fluorescence intensity of LNF was measured in a diluting solvent composed of 2% Tween‐80: phosphate buffer pH 8.0 (20: 80, v/v) using excitation and emission wavelengths of 290 and 450 nm, respectively. The proposed method was fully validated as per the ICH guidelines. The recorded fluorescence intensity of LNF was rectilinear over a concentration range of 0.3–2 μg/ml with a high correlation coefficient (r = 0.9990) and low limits of detection (0.091 μg/ml) and quantitation (0.275 μg/ml). The applicability of the method was extended to study the inherent stability of LNF under different stress degradation conditions including, alkaline, acidic, oxidative, photolytic and thermal degradation. Moreover, the method was utilized to study the kinetics of the alkaline and oxidative degradation of LNF. The pseudo‐first order rate constants and half‐lives were calculated.