Mean Percentage Recoveries Research Articles

Method Development and Validation for Simultaneous Estimation of Glimepiride and Simvastatin by using Reversed Phase High-performance Liquid Chromatography

An analytical method was developed using reverse phase high performance liquid chromatograph (RP-HPLC) system and a C-18 reverse-phase column (Nucleodur C18, 250mm × 4.6mm i.d.,5μ) for simultaneous estimation of simvastatin and glimepiride. Acetonitrile and potassium dihydrogen phosphate buffer pH 4 (75:25, v/v) were used as mobile phase. The flow rate was 1 mL min−1 and the chromatogram of both drugs was detected at wavelength of 232 nm. Method was validated as per ICH Q2 (R1) guidelines. The retention times of glimepiride and simvastatin was found to be 4.726 min and 9.829 min, respectively. Both drugs have shown linearity over the concentration range 2–10μg/mL with r2 of 0.997 for GLM and 0.998 for SIM. The mean percentage recovery of both the drugs was found within 98–102% at all the levels which indicated that the method was accurate. The percentage relative standard deviation was found less than 2% which indicated that method was satisfactorily precised. The LOD and LOQ were found to be 0.24 and 0.73 for simvastatin and 0.32 and 0.96 for glimepiride. The method was found to be robust as there was no significant change in response with variation in pH, flow rate and mobile phase composition. It was concluded that the developed method has passed all the validation tests and can be successfully applied to estimate the presence of both the drugs in bulk as well as in pharmaceutical formulations.

Development and Validation of a Stability indicating RP-HPLC Method for the Estimation of Chlorthalidone and Cilnidipine in Combined Pharmaceutical Dosage Form

A simple and a rapid stability indicating RP-HPLC method has been developed for the estimation of Chlorthalidone and Cilnidipine in their combined dosage form. The separation was achieved on waters C18 column, (100mm x 4.6mm, 2.5μm) using a mobile phase consisting of phase A, (0.1%Formic acid in methanol: Acetonitrile in the ratio 80: 20 v/v) phase B, (10 mM ammonium acetate) in gradient mode, at aflow rate of 1.0mL/min. The wavelength selected for detection was 240 nm and a PDA detector was employed for the detection. The retention time of Chlorthalidone was 6.047±0.2 min and Cilnidipine was 12.642 ±0.2 min. The developed method was validated for specificity, linearity, accuracy precision and robustness. There was no interference seen in the blank at the retention time of the drugs hence the method was specific. The linear regression seen in calibration curve of both the drugs was 0.999. The mean percent recovery of Chlorthalidone and Cilnidipine was within the acceptance criteria. Both the drugs were subjected to a variety of stress environment which included acid, base, peroxide, UV radiations and heat. It was observed that the peaks of the degradants of both the drugs could be well resolved from that of pure drugs. The proposed method can be used for rapid quantification and simultaneous analysis of both drugs from commercial formulations without any interference.

Simultaneous Spectrophotometric Determination of Trigonelline, Diosgenin and Nicotinic Acid in Dosage Forms Prepared from Fenugreek Seed Extract.

Mathematical algorithms offer a useful method for quantitative analysis of compounds in multi-component mixtures to overcome the overlapping problems occurred in UV spectrophotometry. The aim of this study is to develop a method for simultaneous determination of bioactive compounds in herbal dosage forms produced from fenugreek extract. A UV- spectrophotometric method based on mathematical algorithm was used to simultaneous determination of trigonelline (TRG), diosgenin (DI), and nicotinic acid (NA). The maximum absorbance (λmax) was determined to be 232.65 nm, 296.23 nm, and 262.60 nm for TRG, DI, and NA, respectively. The calibration curves showed good linearity for all analytes in the concentration range of 1–20 μg/mL (R2=0.9995, 0.9997, 0.9994 for TRG, DI and NA, respectively). The Intra- and inter-day precisions were in the range of 1.1-10.7% and 1.2-8.2%, respectively. The accuracy of the method was 96.0% for TRG, 92.9% for DI, and 104.2% for NA. The limits of detection (LOD) and quantification (LOQ) were found to be 0.91 and 3.06 µg/mL for TRG, 0.99, and 3.30 µg/mL for DI and 0.33 and 1.10 µg/mL for NA. The validated method was applied for determination of the analytes in the tablet, capsule and thin film dosage forms prepared from the fenugreek seed extract. The mean recovery percentages of the analytes were in the range of 90.0-97.4%, 85.6-105.4%, and 90.0-99.0% for tablet, capsule, and film dosage forms, respectively. Generally, the validated method could be a good candidate for routine spectrophotometric determination of the analytes without any necessity for pre-analysis extraction.

Total mercury bias in soil analysis by CV-AFS: causes, consequences and a simple solution based on sulfhydryl cotton fiber as a clean-up step.

This study examines the matrix effect over the trueness for determining total mercury (THg) using CV-AFS. We demonstrate that matrix interferences in soils and sediment samples cannot be eliminated by acid digestion and establish the use of sulfhydryl cotton fiber (SCF), a malleable, cheap and easy to synthesize fiber, as a mandatory solution capable to overcome this bias. Using the classic CV-AFS approach, an overestimation bias for THg recovery values of >140% in a certified reference material was reported. Interference metals test was conducted, thus discarding any influence of metals in the overestimation bias. Therefore, a clean-up step using SCF was proposed, and tests with synthesized fiber did not present a dispersion of >0.08 ng L-1. Moreover, validation was performed by analyzing three certified reference material and yielding mean recovery percentages of 100% ± 1%. A validated methodology was applied to ten environmental soil samples; THg values obtained varied from 129 to 384 ng g-1. Finally, a comparison between sample results obtained and reference method did not show any significant differences (p > 0.05), thus highlighting the efficacy of SCF-CV-AFS for THg quantification in environmental solid samples.

Conventional and first derivative synchronous spectrofluorimetric methods for the simultaneous determination of cisatracurium and nalbuphine in biological fluids.

Cisatracurium besylate has been determined by fast and highly sensitive spectrofluorimetric method based on measuring the fluorescence intensity of its methanolic solution at 312nm after excitation at 230nm (Method I). The linearity occurred over the concentration range of 10.0-130.0ng/mL with detection limit of 1.07ng/mL. The method was further extended for the determination of the studied drug in spiked human plasma with good percentage recoveries (97.43-103.50%). Cisatracurium is co-administered with nalbuphine during surgery. The simultaneous determination of both drugs was based on synchronous spectrofluorimetric technique. First derivative synchronous spectrofluorimetric amplitude was measured in methanol at Δ λ=60nm and each drug could be estimated at the zero crossing point of the other. Hence, cisatracurium could be measured at 284.6nm while nalbuphine at 276.3nm (Method II). The method was linear over the ranges of 50.0-750.0ng/mL and 0.5-7.0μg/mL with the detection limits of 2.16ng/mL and 0.04μg/mL for cisatracurium and nalbuphine, respectively. The method was further extended for the simultaneous determination of both drugs in spiked human urine with mean percentage recoveries of 99.99±2.06 and 99.53±6.17 for cisatracurium and nalbuphine, respectively. Both methods were validated in agreement with Guidelines adopted by International Council of Harmonization (ICH).

STABILITY-INDICATING RP-UPLC METHOD FOR SIMULTANEOUS DETERMINATION OF DOLUTEGRAVIR AND RILPIVIRINE IN BULK AND PHARMACEUTICAL DOSAGE FORM

Specific stability-indicating reversed-phase ultra-performance liquid chromatography (UPLc) method has been developed and validated for the simultaneous quantification of dolutegravir and rilpivirine in bulk drugs and pharmaceutical dosage forms. the optimized conditions for the developed UPLc method are Sb c8 column (100 x 3 mm, 1.8 mm) maintained at 30°c with mobile phase consisting of 0.1% ortho phosphoric acid and acetonitrile in the ratio 55:45%v/v in isocratic mode at flow rate of 1 mL/min and detection wavelength 260 nm. the retention times of dolutegravir and rilpivirine were found to be 1.25 min, and 1.69 min with linearity in the concentration ranges from 12.5 – 75.0 µg/mL and 6.25 – 37.5 µg/mL, respectively. the mean percentage recoveries of dolutegravir and rilpivirine were found to be 99.04 - 99.79 % and 99.20 - 99.92 %, respectively. the percent relative standard values were less than 2.0 at all the levels, indicating satisfactory accuracy and precisiom. the robustness was found to meet the acceptance criteria. the stress study against qualified working standards of dolutegravir and rilpivirine indicated that the developed UPLc method was stability- indicating, conducted as per ICH requirements.

Analytical performances of a novel point-of-care procalcitonin assay

ObjectivesWe report the analytical performances of a new point-of-care (POC) procalcitonin (PCT) fluorescence immunoassay that uses the AFIAS-6© system from Boditech and its concordance with results of the standard method Kryptor Compact plus from the central laboratory. Designand methods: Analytical performances including imprecision studies, limit of blank (LoB), limit of detection (LoD) and limit of quantification (LOQ) were determined. The method comparison was performed using plasma vs. whole blood for Kryptor CompactPlus© vs. AFIAS-6©, respectively. ResultsThe total imprecision was far from the CV of 4.5% claimed by the manufacturer and close to 10%, for levels of PCT at 0.4 and 8.3 μg/L. The LoD of this novel PCT assay was found to close to the LoD provided by the manufacturer at 0.04 μg/L. The LOQ was higher than that claimed by the manufacturer (0.1 vs 0.002, respectively). The equation of linearity in the lower range was found to be y = 1.056x – 0.039 with r2 = 0.993 with a mean recovery percentage of 86 ± 15%. Correlation studies showed a good correlation between PCT measurements using plasma on Kryptor system and on corresponding whole blood with POC reaching a bias of −0.04 in the range from 0.02 to 2 μg/L. ConclusionThe novel PCT assay on AFIAS-6© is an acceptable POC alternative for the diagnosis and management of sepsis at EDs to improve the flow of patients, as results are consistent with those of the standard PCT Kryptor Compact Plus© assay, despite its higher imprecision.

Fluorescamine-based fluorophore for spectrofluorimetric determination of heptaminol in human plasma; application to spiked human plasma.

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 471 nm after excitation at 393 nm. The linearity of the constructed calibration curve was (75-850 ng/ml) with LOD and LOQ values 23.85 and 72.29 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 invitro study of heptaminol in spiked human plasma with a mean percentage recovery 100.52 ± 1.19% as well as in its tablets with a mean percentage recovery 99.47 ± 1.25%.

Stability indicating RP-HPLC method for simultaneous determination of omeprazole and aspirin in the presence of salicylic acid as degradation product

Reversed-phase high performance liquid chromatographic method (RP-HPLC) was applied for simultaneous determination of aspirin (ASP) and omeprazole (OMP) in presence of salicylic acid (SA) as the most common degradation product of ASP. The drugs were exposed to different stress conditions including acid, alkali, neutral hydrolysis, oxidation, and photodegradation. The separation was carried out on the Thermo Scientific Hypersil ODS column (250 × 4.6 mm, 5 µm) column. Mobile phase composed of acetonitrile: 0.05 M sodium phosphate monobasic buffer with 0.1% TEA (23:77, v/v). The pH of the buffer was adjusted to pH 3.5 with o-phosphoric acid. The flow rate was 1.2 mL/min. The UV detector was set at 230 nm. The retention time of SA, ASP and OMP were 3.51 ± 0.19 min, 4.77 ± 0.23 min, and 16.65 ± 0.34 min, respectively. Linearity was established for in the range of 1–150 µg/mL for ASP, 2–80 µg/mL for OMP and 1–25 µg/mL for SA. The mean percentage recoveries of ASP, OMP and SA were found to be 99.89% ±0.39, 99.76% ±0.38 and 100.28% ±0.18, respectively. The developed method was validated and successfully applied to the simultaneous determination of ASP and OMP in their tablets. The assay results obtained using the developed method were statistically compared to those obtained by the reported RP-HPLC method and good agreement was observed.

In vitro liquid culture and optimization of Steinernema jeffreyense using shake flasks

Entomopathogenic nematodes (EPNs) of the families Heterorhabditidae and Steinernematidae are efficient biological control agents against important insect pests. In vitro liquid culture production technology is a key factor in the success of implementing EPNs as a biological control agent. One of the first steps of in vitro mass culture is to use shake flasks to obtain nematode inoculum for optimising and upscaling to desktop and industrial fermenters. This study was the first attempt on the in vitro liquid mass culture of a local South African isolate, Steinernema jeffreyense, in 250 ml Erlenmeyer flasks, together with their mutualistic bacteria, Xenorhabdus khoisanae. After the successful in vitro production of S. jeffreyense-inoculum, different parameters for optimizing infective juvenile (IJ) recovery (developmental step when the IJ moult to initiate the life cycle) and yield, were investigated. This includes the effect of the volume of liquid medium in the flasks, two different orbital shakers setups and the initial IJ inoculum density. With 30 ml of liquid medium the mean percentage recovery of IJ after six days was 86%, with a yield of 121,833 IJ ml−1 after 14 days, in comparison to 75% and 99,875 IJs ml−1 respectively when 50 ml of liquid medium was used. No significant difference was found between IJ recovery and yield, using different orbital shakers setups. Among the three inoculum concentrations tested (1000, 2000 and 3000 IJ ml−1), the lowest concentration gave the highest IJ recovery and yield. Pathogenicity of IJs cultured in vitro was higher than those cultured in vivo.

Optimization and validation of a new chromatographic method for the assay of veterinary formulation

New, validated and accurate reversed phase HPLC method with UV detection has been established for simultaneous determination of a veterinary binary mixture of doxycycline hydrochloride (DOX) and tylosin tartrate (TYT). The stationary phase was ACE- 126-2546 AQ C-18 (250 × 4.6 mm i.d., 5 μm particle size) column at 25 °C, in an isocratic mode, using mobile phase containing a mixture of methanol: acetonitrile: distilled water in the ratio of 60:20:20 (v:v:v), with 0.01% trichloroacetic acid at the flow rate of 0.8 mL/min and UV detection was performed at 270 nm. The retention times were 4.02±0.01 and 5.62±0.01 mins for DOX and TYT, respectively. Selective determination of the cited veterinary drugs has been developed in their formulation. The method was found to be linear over 1-50 µg/mL for DOX and TYT with mean percentage recoveries 99.62±1.220 and 100.09±1.104%. The method was proven to be accurate, precise and specific. The obtained results were statistically compared with those of the official and reported methods; using Student’s t test, F test and one-way ANOVA, showing no significant difference with high accuracy. Specificity of the applied method was assessed by analysing the laboratory-prepared mixtures and their combined dosage form. The developed method was confirmed according to ICH guidelines. The validated method can be considered as alternative and basic method for the routine determination of this fixed dose combination with minimum sample preparation.

DETERMINATION OF MEFENAMIC ACID IN A TOPICAL EMULGEL BY A VALIDATED HPLC METHOD

Objective: The present study is aimed to develop and validate a simple, precise and accurate high-performance liquid chromatography (HPLC) method, according to ASEAN guideline for the validation of the analytical procedure, for the determination of mefenamic acid in a topical emulgel preparation.
 Methods: An emulgel of 1 % mefenamic acid was prepared using carbopol 940 as a gelling agent and cremophor EL as an emulsifying agent. It was diluted with ethanol to make a sample concentration of 200 mg/ml. The method used a C18 column (5 µm; 250 x 4.6 mm) with the mobile phase, consisting of acetonitrile, acetic acid, and water in a ratio of 75:1:24. The column was maintained at 25 °C. The flow rate was 1 ml/min and the injection volume was 10 ml. The peak response was monitored by UV at 282 nm. It was validated for specificity, range, linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ). In addition, forced degradation (hydrolysis, oxidation and dry heat) was performed to determine the capability of the proposed method to analyze the chemical stability of the drug samples during storage.
 Results: The method was specific to the drug while other excipients did not interfere with the quantitation of mefenamic acid. It was linear in the concentration range of 1.29 to 806 mg/ml. LOD and LOQ were 4.88 and 14.78 mg/ml, respectively. Accuracy of the method was demonstrated by recovery experiments on the synthetic mixture method and the mean percent recovery was 101.10±1.56. Repeatability and intermediate precision were rugged with %RSD values of 1.30 and 1.07, respectively. The method could separate mefenamic acid from other degradation products of forced degradation.
 Conclusion: The HPLC method presented herein is simple, accurate, sensitive and reproducible for the determination of mefenamic acid in an emulgel. It is served as a stability-indicating method and can be used for the analysis of the drug during product development and stability studies.

HPTLC Method for Determination of R, R-Glycopyrronium Bromide and its Related Impurities.

An innovative high-performance thin layer chromatographic (HPTLC) method was designed, optimized and validated for the quantification of R, R-glycopyrronium bromide (GLY) and its related impurities in drug substance and drug product. Separation was performed on HPTLC plates pre-coated with silica gel 60F254 by dichloromethane:methanol:formic acid (10:0.5:0.5, v/v/v) as a developing system. GLY and its related impurities namely, glycopyrronium impurity G and glycopyrronium impurity J, were separated giving compact well-resolved spots with significant retardation factor (Rf) values of 0.17±0.02, 0.34±0.02 and 0.69±0.02, respectively. Quantification was done at 220nm in the ranges of 0.3-10 and 0.2-4.0μg/spot with limits of detection and quantification of 0.1, 0.3 and 0.05, 0.2μg/spot for GLY and its related impurities, respectively. Good accuracy was obtained with mean percentage recovery of 99.48±1.36, 100.04±1.32 and 99.61±0.80 and R2≥0.9968 for GLY and its impurities, consecutively. Validation parameters were presented according to the International Conference on Harmonization. The method was used to investigate impurity profile of GLY in drug substance and drug product and could be applied in routine analysis of the drug. Comparison between the developed method and the reported method revealed no statistical difference.

Functional Recovery via Preregenerated Nerve Graft in Rat Sciatic Nerve Injury Model

Abstract INTRODUCTION Patients who have experienced major tissue loss with peripheral nerve injury (eg, limb amputation) may be offered composite tissue allotransplantation (CTA). The return of sensory ability and cosmetic component of CTAs make them an attractive alternative to prosthetic devices. Unfortunately, robust reinnervation especially over great distances remains an issue for hand allotransplants. In this study, we introduce a preregenerated nerve graft to shorten the distance and therefore time to terminal tissue reinnervation, which could improve the utility of CTAs. METHODS A total of 18 rats weighing 250 to 300 gm each were randomized into 1 of 3 groups: baseline, fresh, or preregenerated. The baseline group underwent sham surgery to obtain baseline functional data. The fresh and preregenerated groups both underwent grafting of the sciatic nerve but the preregenerated group utilized 8-wk preregenerated grafts. At postperative week 8 from distal neurorrhaphy, both groups underwent terminal functional testing via EMG and evoked muscle force. RESULTS The preregenerated group had significantly greater mean EMG (P < .05) and maximum tetanic muscle force values (P < .05) than the fresh group. Mean percent recovery in EMG for the fresh group was 21.95% compared with 81.79% in the preregenerated group. Mean percent recovery in muscle force was 9.46% and 33.15%, respectively. CONCLUSION The results of this study provide a novel approach to enhance final functional recovery after peripheral nerve injury. The current practice of constructing a nerve stump may be improved by grafting a nerve segment at the time of injury and allowing it to preregenerate into local musculature so that if a CTA is later performed, an expedited and more robust reinnervation could be accomplished.

Mixed micellar liquid chromatographic method for simultaneous determination of norfloxacin and tinidazole in pharmaceutical tablets

A new green mixed micellar liquid chromatographic method was developed, validated, and successfully applied for the determination of norfloxacin and tinidazole in bulk and in pharmaceutical dosage form. The developed method used the anionic surfactant; sodium dodecyl sulphate and the neutral surfactant polyoxyethylene (23) lauryl ether without need of organic solvent in the mobile phase. Optimization of the chromatographic condition was performed by 23 full factorial design. Chromatographic separation and quantitation was performed on ODS Hypersil® column (250 × 4.6 mm, 5 μm). Mixed-micellar mobile phase consisted of 0.14 M SDS, 0.03 M Brij-35 and 0.3% triethylamine. pH of the mobile phase was adjusted to 2.9 by orthophosphoric acid. The flow rate was 1 mL/min. The injection volume was 20 μL. UV detection was performed at 313 nm. Linearity ranges were found to be 2–10 μg/mL and 2–12 μg/mL for norfloxacin and tinidazole, respectively. The developed method was validated and successfully applied to the simultaneous determination of both drugs in their tablet. The mean percentage recovery ± S.D. values were found to be 97.745 ± 0.664 and 100.441 ± 1.379 for norfloxacin and tinidazole, respectively. The assay results obtained were statistically compared to those obtained by a reported method and good agreement was observed. The present method offers significant advantages compared to previously reported chromatographic methods concerning column efficiency, selectivity and resolution. The green property of the method was evaluated. The method is verified to obtain excellent greenness. The calculated analytical Eco-Scale of the method was also compared to those of other reported liquid chromatographic methods.

Factor Optimization Study to Develop and Validate a Reversed-Phase Thin-Layer Chromatography Method for the Determination of Trimetazidine Dihydrochloride and Its Reported Impurities in Pharmaceuticals

A validated reversed-phase thin-layer chromatography (RP-TLC)—densitometry method was developed and optimized for the determination of trimetazidine dihydrochloride (TMZ) and its potential impurities listed in the British Pharmacopoeia, namely, piperazinecarboxaldehyde (Y-145), trimethoxybenzyl alcohol (Y-235), and trimethoxybenzaldehyde (Y-234). Chromatographic separation was performed on aluminum plates pre-coated with silica gel 60 RP-18F using a mixture of acetonitrile—methanol—0.1% aqueous ortho-phosphoric acid pH 6.2 (4.5:4.5:1, V/V) as the developing system. Different factors affecting resolution were studied and optimized. Successful resolution was observed with significant difference in the RF values of 0.21 ± 0.02, 0.35 ± 0.02, 0.5 ± 0.02, and 0.85± 0.02 for TMZ and its impurities, respectively. Densitometric measurement was done at 215 nm over the range of 0.05–10, 0.05–1.1, 0.05–1.2, and 0.04–1.0 µg per spot with lower limits of detection (LOD) and quantification (LOQ) of 20 and 50 ng per spot for TMZ, Y-145, and Y-235, respectively, and 15 and 40 ng per spot for Y-234 impurity. Good accuracy was obtained with mean percentage recoveries of 99.55 ± 1.06, 100.50 ± 1.16, 100.07 ± 1.24, and 99.41 ± 1.11 for TMZ and its impurities, respectively. The developed method was used to investigate the impurity profile of TMZ in drug substance and different products and it was validated as per the International Conference on Harmonisation (ICH) guidelines.

Recovery function of somatosensory evoked potentials in juvenile myoclonic epilepsy*

Objective: We analysed the recovery function of somatosensory evoked potentials (SEPs) in juvenile myoclonic epilepsy (JME) patients. We hypothesized that there may be disinhibition in the recovery of SEPs at 20–100 ms intervals in JME patients.Methods: We recorded SEPs and SEP recovery in 19 consecutive patients with JME admitted for a routine follow-up examination, and in a control group composed of 13 healthy subjects who were similar to the patient group regarding age and sex. The recovery function of SEPs was examined using paired stimuli at 30, 40, 60, and 100 ms intervals.Results: The amplitudes of N20-P25 and P25-N33 components were higher in patients with JME. Ten patients had high-amplitude SEPs. By paired stimulation, there was inhibition of SEPs in both groups. The mean recovery percentages of N20-P25 and P25-N33 components at 30, 40, 60, and 100 ms were not different between healthy subjects and patients with JME.Conclusions: The recovery function of SEP is normal in JME even in the presence of high-amplitude SEPs.

Development and Validation of HPLC Fluorescence and UPLC/DAD Stability-Indicating Methods for Determination of Hepatitis C Antiviral Agent Daclatasvir.

Background: Few stability-indicating chromatographic methods were published for determination of daclatasvir. All used UV detection. Objective: This work aimed to develop rapid, specific, and novel stability-indicating methods using HPLC with fluorescence detection and ultra performance liquid chromatography (UPLC) with UV detection for the determination of daclatasvir in bulk powder and in its dosage form. Methods: The drug was subjected to hydrolysis (acidic and alkaline) as per International Conference on Harmonization (ICH) guidelines. The fragmentation pattern of the drug was studied using LC-MS. Separation was carried out first by HPLC using Thermo BDS Hypersil Phenyl (300 mm × 4 mm, 5 μm) column and a mobile phase consisting of ammonium phosphate buffer (0.02 M) pH 3-methanol (40+60, v/v) at flow rate 1 mL/min. Quantitation was achieved by fluorescence detection at 305 and 457 nm for excitation and emission, respectively. The second method used UPLC equipped with diode-array detector. Acquity BEH C18 (50 mm × 2.1 mm, 1.7 μm) column was used with the same mobile phase at flow rate 0.4 mL/min and detection wavelength at 305 nm. ICH guidelines were used for validation. Results: The mean percentage recovery ± SD values for tablet assay were found to be 101.12 ± 0.655 and 99.78 ± 0.632 by HPLC and UPLC methods, respectively. The assay results showed a good agreement with the reported method. Conclusions: The developed HPLC and UPLC methods provide simple, accurate, and reproducible analysis of daclatasvir without interference from degradates. Highlights: This is the first research using fluorescence detection in a stability-indicating chromatographic method for determination of daclatasvir.

Assessment of the greenness of spectrophotometric and micellar liquid chromatographic methods using two approaches: Application to pharmaceutical analysis of hydrochlorothiazide and telmisartan

Recently the focus of analytical community is to eliminate or reduce the usage of hazardous chemicals; consequently, two ecofriendly, rapid, selective and cost-effective methods are developed for the simultaneous determination of hydrochlorothiazide (HCZ) and telmisartan (TEL). The first method is based on micellar high pressure liquid chromatography using an ecofriendly micellar mobile phase consists of 0.06 M sodium dodecyl sulfate (SDS), 12% v/v n-propanol and 0.3% v/v triethylamine (TEA) at pH 6.0 pumped at a flow rate of 2 mL/min. Rivaroxaban (RIV) is used as an internal standard (I.S). The method gives linear correlation over concentration ranges of 0.8–20.0 μg/mL and 1.0–20.0 μg/mL for HCZ and TEL, respectively. The mean percentage recovery for HCZ is 100.60 ± 0.92 while for TEL is 100.07 ± 1.16. The second method is based on absorbance ratio (Q-absorbance) technique which depends on measuring the absorbance at two certain wavelengths, one of which is the isoabsorptive point and the other is the λmax of one of the two drugs. Methanolic solutions of HCZ and TEL show their isoabsorptive points at 264.6 nm and 277.3 nm while the λmax of HCZ in methanol is 270 nm. The two drugs obey Beer-Lambert law over the concentration ranges of 1.0–20.0 μg/mL for HCZ and TEL, respectively. For HCZ, the mean percentage recovery is 99.86 ± 0.87 while for TEL it is 99.83 ± 0.94. The spectrophotometric method does not need the addition of any chemical reagents and is carried out without heating or extraction steps. Both methods are successfully applied to the analysis of both drugs in their combined tablets. The results are validated according to official guidelines. Statistical evaluation of the results achieved by the developed and comparison official methods confirms the high accuracy and precision of the developed methods as indicated by student t-test and variance ratio F-test, respectively. Moreover, the greenness of the developed methods is assessed using suitable analytical Eco-Scale and greenness profile, and comparison with the previously published methods has been carried out to indicate priority of the proposed procedures.

Computational design of molecularly imprinted polymer for solid phase extraction of moxifloxacin hydrochloride from Avalox® tablets and spiked human urine samples

In this paper, moxifloxacin hydrochloride molecularly imprinted polymer was used as a selective sorbent for solid phase extraction of moxifloxacin hydrochloride from different matrices. First a computational study was conducted for confirming the optimum composition of the polymer to be used as the sorbent. Then, different parameters were studied during optimization of the solid phase extraction procedures, including; the amount of sorbent, cartridge capacity, composition of the washing and elution solvent. A new UPLC-MS/MS method was developed for the quantification purposes. The newly developed method was validated according to the ICH guidelines in terms of; linearity, limit of detection, limit of quantification, precision and accuracy. The optimized solid phase extraction method coupled with the validated UPLC-MS/MS method was used for the determination of moxifloxacin HCl in Avalox® tablets (400 mg/tablet) and in spiked human urine samples. The mean percentage recoveries ranged from 82.21 to 109.36% with percentage relative standard deviation value ranging from 0.0001 to 10.88%.