HPLC Separation of Nimesulide and Five Impurities using a Narrow-Bore Monolithic Column: Application to Photo-Degradation Studies

Nimesulide and its fine impurities were separated using the new narrow-bore FastGradient monolithic column (50 × 2.0 mm i.d.). The mobile phase was a mixture of NH₄H₂PO₄ (10 mmol L^?1, pH 7)/ACN (69:31 v/v) and UV detection was carried out at 230 nm. Depending on the flow rate of the mobile phase (0.5?C1.0 mL min^?1), efficient separation of the six compounds could be achieved within 5?C10 min. The developed HPLC method was validated in terms of linearity, limits of detection and quantitation, precision, selectivity, and accuracy. Application to the photo-stability of nimesulide revealed a ca. 50% degradation of the active pharmaceutical ingredient within 24 h. Impurity D was also identified at a maximum level of 0.46% (24 h).     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min⁻¹ for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.

     

High-Performance Liquid Chromatographic Method for the Determination of Nimesulide in Pharmaceutical Preparations

A simple, rapid, and precise high-performance liquid chromatographic method for the determination of nimesulide in pharmaceutical preparations was proposed using Ibuprofen as an internal standard. The separation was performed on a CLC C₁₈ (5 m, 25 cm × 4.6 mm i.d.) column with a mobile phase consisting of an acetonitrile–0.05 M KH₂PO₄ buffer mixture of pH 7.00 (55 : 45, v/v). The detection was carried out at 230 nm and the linearity range was found to be 0.5–100 g/mL. The method has been applied successfully to the determination of nimesulide in pharmaceutical formulations. The recovery values were found to be in the range of 99.23–100.13% with RSD values of less than 0.97%.     

Nimesulide/cyclodextrin/PEG 6000 ternary complexes: physico-chemical characterization, dissolution studies and bioavailability in rats

Purpose Ternary solid dispersions were prepared in order to estimate the effect of a double hydrophilization by cyclodextrins and PEG 6000 on nimesulide apparent characteristics. Ternary solid dispersions of nimesulide, cyclodextrins and PEG 6000 were characterized using DSC, FT-IR, dissolution studies and evaluating the bioavailability in rats. Methods Ternary solid dispersions were prepared either using native powders or using a preformed inclusion complex of nimesulide and cyclodextrin. Inclusion complexes and pure drug were used as references. Circulating nimesulide was measured out in rat plasma after orally administration of our different products (ternary solid dispersions, inclusion complexes and pure drug). Results An improvement of the nimesulide dissolution rate was obtained with inclusion complexes and ternary solid dispersion. In rat plasma, inclusion complexes and ternary solid dispersion improved T _max. Conclusions A second hydrophilization of inclusion complexes by PEG 6000 does not allow to achieve better results concerning nimesulide concentration in rat plasma or in dissolution studies than with inclusion complexes alone.     

Determination of nimesulide in pharmaceutical and biological samples by a spectrophotometric method assisted with the partial least square method

A simple and sensitive spectrophotometric method for the determination of nimesulide in bulk, in pharmaceutical dosage form, and in biological fluids was developed. The method is based on the reduction of the nitro group of nimesulide by zinc and hydrochloric acid followed by diazotization, and coupling with orcinol in basic medium to form a stable chromophore, which absorbs at 465 nm. The method showed a good linearity in the range 0.4–4.0 μg mL^?1. Partial least square modeling as a powerful multivariate statistical tool is also applied, compiled, and compared for determination of nimesulide. The experimental matrix for the partial least square calibration method was designed with 24 samples. The cross-validation was used for selecting the number of factors. The root mean square error prediction (RMSEP) and the relative error of prediction (REP %) were 0.089 and 3.95, respectively. The developed method is free from the interference of common excipients used in pharmaceutical dosages. The method was also used for the determination of nimesulide in pharmaceutical dosages as well as in human serum and urine samples.     

Development and validation of an HPLC method for tetracycline‐related USP monographs

A novel reversed‐phase HPLC method was developed and validated for the assay of tetracycline hydrochloride and the limit of 4‐epianhydrotetracycline hydrochloride impurity in tetracycline hydrochloride commercial bulk and pharmaceutical products. The method employed L1 (3 µm, 150 × 4.6 mm) columns, a mobile phase of 0.1% phosphoric acid and acetonitrile at a flow rate of 1.0 mL/min, and detection at 280 nm. The separation was performed in HPLC gradient mode. Forced degradation studies showed that tetracycline eluted as a spectrally pure peak and was well resolved from its degradation products. The fast degradation of tetracycline hydrochloride and 4‐epianhydrotetracycline hydrochloride in solution was retarded by controlling the autosampler temperature at 4 °C and using 0.1% H₃PO₄ as diluent. The robustness of the method was tested starting with the maximum variations allowed in the US Pharmacopeia (USP) general chapter Chromatography <621>. The method was linear over the range 80–120% of the assay concentration (0.1 mg/mL) for tetracycline hydrochloride and 50–150% of the acceptance criteria specified in the individual USP monographs for 4‐epianhydrotetracycline hydrochloride. The limit of quantification for 4‐epianhydrotetracycline hydrochloride was 0.1 µg/mL, 20 times lower than the acceptance criteria. The method was specific, precise, accurate and robust. Copyright © 2014 John Wiley & Sons, Ltd.     

A Simple Strategy for Simultaneous Determination of Paracetamol and Caffeine Using Flow Injection Analysis with Multiple Pulse Amperometric Detection

In this work, we report a simple and novel strategy for simultaneous analysis using flow injection analysis with multiple pulse amperometric (FIA‐MPA) detection. The proposed strategy was successfully used for simultaneous determination of paracetamol and caffeine (model analytes) in pharmaceutical formulations. A sequence of potential pulses (waveform) was selected in such a way that PA is selectively oxidized at E₁ (+1.20 V/50 ms) and both compounds (PA+CA) are simultaneously oxidized at E₂ (+1.55 V/50 ms); hence, current subtraction (using a correction factor) can be used for the selective determination of CA. The proposed FIA method is simple, cheap, fast (140 injections h^?1), and present selectivity for the determination of both compounds in pharmaceutical samples, with results similar to those obtained by HPLC at a 95 % confidence level.     

Simultaneous determination of five coumarins in Angelicae dahuricae Radix by HPLC/UV and LC‐ESI‐MS/MS

Rapid, simple and reliable HPLC/UV and LC‐ESI‐MS/MS methods for the simultaneous determination of five active coumarins of Angelicae dahuricae Radix, byakangelicol (1), oxypeucedanin (2), imperatorin (3), phellopterin (4) and isoimperatorin (5) were developed and validated. The separation condition for HPLC/UV was optimized using a Develosil RPAQUEOUS C₃₀ column using 70% acetonitrile in water as the mobile phase. This HPLC/UV method was successful for providing the baseline separation of the five coumarins with no interfering peaks detected in the 70% ethanol extract of Angelicae dahuricae Radix. The specific determination of the five coumarins was also accomplished by a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source (LC‐ESI‐MS/MS). Multiple reaction monitoring (MRM) in the positive mode was used to enhance the selectivity of detection. The LC‐ESI‐MS/MS methods were successfully applied for the determination of the five major coumarins in Angelicae dahuricae Radix. These HPLC/UV and LC‐ESI‐MS/MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). Taken together, the shorter analysis time involved makes these HPLC/UV and LC‐ESI‐MS/MS methods valuable for the commercial quality control of Angelicae dahuricae Radix extracts and its pharmaceutical preparations. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of Nimesulide by Ion Pair High-Performance Liquid Chromatography Using Tetrabutylammonium as the Counterion

A new method for nimesulide was developed using ion-pair reversed phase liquid chromatography and tetrabutylammonium hydrogen sulfate as the ion-pairing reagent. The influence of the ion pair forming reagent concentration, pH, and mobile phase composition on the retention time of nimesulide were studied. The optimum experimental conditions included a C18 column, a mobile phase of a 50/50 (v/v) mixture of acetonitrile and 15 mM phosphate buffer (pH 8.00) containing 6 mM tetrabutylammonium hydrogen sulfate, 25°C, isocratic elution, a flow rate of 1 mL/min, a run time of 10 minutes, and photodiode array detection at 404 nm. From the analysis of the results, the mechanism for the separation of nimesulide was also established. The retention time for nimesulide was 4.76 ± 0.05 min. The method was linear between concentrations of 9 µg/mL to 64 µg/mL, with limits of detection and quantification of 1.111 µg/mL and 3.390 µg/mL, respectively. The method is simple, rapid, accurate, and precise, and successfully applied for the determination of nimesulide in pharmaceutical products.     

Simultaneous Determination Baicalin and Forsythin in Shuanghuanglian Oral Liquid by HPLC/DAD and LC‐ESI‐MS

Rapid, simple and reliable HPLC/DAD and LC‐ESI‐MS methods for the simultaneous determination of baicalin and forsythin in the traditional Chinese medicinal preparation Shuanghuanglian oral liquid were described and validated. The separation condition for HPLC/DAD was optimized using a BDS hypersil C18 column (Thermo, 2.1 × 150 mm, particle size 5 μm) by gradient elution using methanol‐0.2 % ammonium acetate as the mobile phase. The suitable detection wavelength was set at 277 nm for the quantitative analysis of baicalin and forsythin in this method. Some operational parameters of the ESI interface were optimized, negative m/z 445[M?H]^? for baicalin and negative m/z 593[M+CH₃COO]^? for forsythin, positive m/z 447[M+H]⁺ for baicalin and positive m/z 552[M+NH 4]⁺ for forsythin, respectively. These HPLC/DAD and LC‐ESI‐MS methods were validated in terms of recovery, linearity, accuracy and precision (intra‐ and inter‐day validation). These methods can be used as a complementary method for the commercial quality control of Shuanghuanglian oral liquid and its pharmaceutical preparations.     

Stability-Indicating Chromatographic Methods for Simultaneous Determination of Mosapride and Pantoprazole in Pharmaceutical Dosage Form and Plasma Samples

Simple, sensitive, selective, precise, and stability-indicating thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods for the determination of mosapride and pantoprazole in pharmaceutical tablets were developed and validated as per the International Conference on Harmonization guidelines. The TLC method employs aluminum TLC plates precoated with silica gel 60F₂₅₄ as the stationary phase and ethyl acetate/methanol/toluene (4:1:2, v/v/v) as the mobile phase to give compact spots for mosapride (R _f 0.73) and pantoprazole (R _f 0.45) separated from their degradation products; the chromatogram was scanned at 276 nm. The HPLC method utilizes a C18 column and a mobile phase consisting of acetonitrile/methanol/20 mM ammonium acetate (4:2:4, v/v/v) at a flow rate of 1.0 mL min^?1 for the separation of mosapride (t _R 11.4) and pantoprazole (t _R 4.4) from their degradation products. Quantitation was achieved with UV detection at 280 nm. The same HPLC method was successfully used in performing calibrations in lower concentration ranges for both drugs in human plasma using ezetimibe as internal standard. The methods were validated in terms of accuracy, precision, linearity, limits of detection, and limits of quantification. Mosapride and pantoprazole were exposed to acid hydrolysis and then analyzed by the proposed methods. As the methods could effectively separate the drugs from their degradation products, these techniques can be employed as stability-indicating methods that have been successively applied to pharmaceutical formulations without interference from the excipients. Moreover the HPLC method was successfully used in the determination of both drugs in spiked human plasma.     

Determination and Validation of an LC Method for Fluvoxamine in Tablets

A new, simple, rapid and specific reversed-phase high-performance liquid chromatography (HPLC) method was developed and validated for the determination of fluvoxamine in pharmaceutical dosage forms. The HPLC separation was achieved on a C₁₈ μ-Bondapack column (250 mm × 4.6 mm) using a mobile phase of acetonitrile–water (80:20, v/v) at a flow rate of 1 mL min⁻¹. Proposed method is based on the derivatization of fluvoxamine with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS) in borate buffer of pH 8.5 to yield a orange product. The HPLC method is based on measurement of the derivatized product using UV-visible absorbance detection at 450 nm. The method was validated for specificity, linearity, precision, accuracy, robustness. The degree of linearity of the calibration curves, the percent recoveries of fluvoxamine, the limit of detection and quantification, for the HPLC method were determined. The assay was linear over the concentration range of 45–145 ng mL⁻¹ (r = 0.9999). Limit of detection and quantification for fluvoxamine were 15 and 50 ng mL⁻¹, respectively. The results of the developed procedure (proposed method) for fluvoxamine content in tablets were compared with those by the official method. The method was found to be simple, specific, precise, accurate, reproducible and robust.     

Development and Validation of a Liquid Chromatographic Method for the Determination of Leflunomide: Application to in vitro Drug Metal Interactions

Leflunomide is a leading drug for the treatment of rheumatoid arthritis. The principle aim of this study was to develop and validate an RP‐HPLC method for the determination of leflunomide in bulk and pharmaceutical dosage form using diclofenac sodium as an internal standard. For this purpose, chromatography was accomplished on a Purospher Start, C₁₈ (5 (m, 12.5 cm×0.46 cm) column at ambient temperature. Methanol:water (80:20, V/V) solvent system was selected as mobile phase, the pH of which was adjusted to 3.4 by ortho‐phosphoric acid and delivered at a flow rate of 1.2 mL·min⁻¹. Seperation of leflunomide and diclofenac sodium was carried out on a Purospher Start, C₁₈ equipped with a UV‐visible detector at 248 nm. The suitability of the method for the quantitative determination of the drugs is proven by validation in accordance with the requirements laid down by the International Conference on Harmonization (ICH) guidelines. The method was accurate (99.55%–100.03%), specific, linear (R²>0.999) and precise (intra‐day precision 0.023%–0.93% and inter‐day precision 0.26%–0.944%) in the range of 0.5–20 (g·mL⁻¹. The minimum limit of detection and quantification in pharmaceutical formulation were 0.05 and 0.15 (g·mL⁻¹, respectively. Thus the proposed method is simple, accurate, reproducible and suitable for the routine analysis of leflunomide in pharmaceutical formulations and was applied to study in vitro drug‐metal interactions.     

A tolbutamide–metformin salt based on antidiabetic drug combinations: synthesis,crystal structure analysis and pharmaceutical properties

A drug–drug anhydrous pharmaceutical salt containing tolbutamide {systematic name: 3‐butyl‐1‐[(4‐methylbenzene)sulfonyl]urea, TOL, C₁₂H₁₈N₂O₃S} and metformin (systematic name: 1‐carbamimidamido‐N,N‐dimethylmethanimidamide, MET, C₄H₁₁N₅) was created based on antidiabetic drug combinations to overcome the poor pharmaceutical properties of the parent drugs. Proton transfer and the proportion of the two components were confirmed by ¹H NMR spectroscopy and single‐crystal X‐ray diffraction analysis. Comprehensive characterization of the new pharmaceutical salt crystal, 2‐[(dimethylamino)(iminiumyl)methyl]guanidine (butylcarbamoyl)[(4‐methylbenzene)sulfonyl]azanide, C₄H₁₂N₅⁺·C₁₂H₁₇N₂O₃S^?, was performed and showed enhancement of the pharmaceutical properties, such as lower hygroscopicity and greater accelerated stability than the parent drug MET, and higher solubility and dissolution rate than TOL. The property alterations were correlated with the crystal packing features and potential hydrogen‐bonding sites through observed changes in the crystal structures.     

A validated HPLC method for determination of Artesunate in bulk and tablet formulation

The simple, accurate and precise HPLC method for determination of Artesunate in bulk and tablet dosage form has been developed. Quantitation of drug was carried out on Jasco HPLC system with HiQ-SiL C₈ column (250 mm × 4.6 mm i.d.), using acetonitrile: 1 M sodium acetate buffer (pH 3 adjusted with o-phosphoric acid) in the ratio 70: 30 as mobile phase. Method was developed using Artemether as internal standard and UV detector set at 220 nm. Linear concentration range was found to be 250–2500 μg/mL. The method has been successfully applied to the analysis of drugs in bulk and pharmaceutical formulation. The method was validated with respect to linearity, precision and accuracy as per the International Conference on Harmonisation guidelines.     

Monitoring of the degradation kinetics of diatrizoate sodium to its cytotoxic degradant using a stability‐indicating high‐performance liquid chromatographic method

The X‐ray diagnostic agent sodium diatrizoate (DTA) was studied for chemical degradation. The 3,5‐diamino derivative was found to be the alkaline and acidic degradation product. The 3,5‐diamino degradate is also the synthetic precursor of DTA and it is proved to have cytotoxic and mutagenic effects. A sensitive, selective and precise high‐performance liquid chromatographic stability‐indicating method for the determination of DTA in the presence of its acidic degradation product and in pharmaceutical formulation was developed and validated. Owing to the high toxicity of the degradation product, the kinetics of the acidic degradation process was monitored by the developed RP‐HPLC method. The reaction was found to follow pseudo‐first order kinetics. The kinetic parameters such as rate constant (K ) and half‐life (t _½) were calculated under different temperatures and acid concentrations; activation energy was estimated from the Arrhenius plot. The developed RP‐HPLC method depends on isocratic elution of a mobile phase composed of methanol–water (25:75 v /v; pH adjusted with phosphoric acid), and UV detection at 238 nm. The method showed good linearity over a concentration range of 2–100 μg/mL with mean percentage recovery of 100.04 ± 1.07. The selectivity of the proposed method was tested using laboratory‐prepared mixtures. The proposed method has been successfully applied to the analysis of DTA in pharmaceutical dosage forms without interference from other dosage form additives and the results were statistically compared with the official USP method. Validation of the proposed method was performed according to International Conference on Harmonization guidelines.     

Determination of the design space of the HPLC analysis of water‐soluble vitamins

Analysis of water‐soluble vitamins has been tremendously approached through the last decades. A multitude of HPLC methods have been reported with a variety of advantages/shortcomings, yet, the design space of HPLC analysis of these vitamins was not defined in any of these reports. As per the food and drug administration (FDA), implementing the quality by design approach for the analysis of commercially available mixtures is hypothesized to enhance the pharmaceutical industry via facilitating the process of analytical method development and approval. This work illustrates a multifactorial optimization of three measured plus seven calculated influential HPLC parameters on the analysis of a mixture containing seven common water‐soluble vitamins (B₁, B₂, B₆, B_12, C, PABA, and PP). These three measured parameters are gradient time, temperature, and ternary eluent composition (B1/B2) and the seven calculated parameters are flow rate, column length, column internal diameter, dwell volume, extracolumn volume, %B (start), and %B (end). The design is based on 12 experiments in which, examining of the multifactorial effects of these 3 + 7 parameters on the critical resolution and selectivity, was carried out by systematical variation of all these parameters simultaneously. The 12 basic runs were based on two different gradient time each at two different temperatures, repeated at three different ternary eluent compositions (methanol or acetonitrile or a mixture of both). Multidimensional robust regions of high critical R_s were defined and graphically verified. The optimum method was selected based on the best resolution separation in the shortest run time for a synthetic mixture, followed by application on two pharmaceutical preparations available in the market. The predicted retention times of all peaks were found to be in good match with the virtual ones. In conclusion, the presented report offers an accurate determination of the design space for critical resolution in the analysis of water‐soluble vitamins by HPLC, which would help the regulatory authorities to judge the validity of presented analytical methods for approval.     

Use of Multiplexed CE for Pharmaceutical Analysis

This report describes the evaluation of a multiplexed, 96-capillary array instrument for analytical performance with a view to implementation for routine, high-throughput use. The need for higher throughput analytical capability for today’s busy pharmaceutical laboratory continues to rise. The use of multiplexed capillary electrophoresis is discussed, and the 96 multiplexed capillary array instrument with UV absorbance detection was used to perform high throughput pharmaceutical analysis. Pharmaceutical product assay, pK _a and log P determinations were achieved. Reproducibility of relative migration time and relative peak area between capillaries was found to be acceptable. The multiplexed instrument offered equivalent performance for tablet assay to a conventional CE instrument. Using the multiplexed CE, pK _a values approaching those of the literature values were obtained for a range of drugs. A microemulsion electrokinetic chromatography (MEEKC) method was successfully used without resulting in excessive operating current. The use of multiplexed CE for high throughput analysis has been shown to be a highly viable alternative to HPLC and other conventional analytical techniques.Presented at: CE in the Biotechnology & Pharmaceutical Industries: 7th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, Montreal, Canada, August 12–16, 2005.     

Konzentrationen von Isomeren des Typs P m As4−m X 3 in P4S3-As4S3- und P4Se3-As4Se3-Mischungen

The reactions of P₄S₃ with As₄S₃ and of P₄Se₃ with As₄Se₃ in the molten state yields molecules of the type P _m As_4–m S₃ and P _m As_4–m Se₃, respectively. A method was developed to separate the different components by the HPLC technique, and to determine their concentrations. The identification of the isomers in the HPLC pattern was achieved with the aid of the LC-MS method. In the selenium system, the distribution of the different species is statistical. In the system P₄S₃-As₄S₃, the formation of PAs₃S₃ with one phosphorus atom in the apical position is favoured.

     

The quantitation of butyltin and cyclohexyltin compounds in the marine environment of British Columbia

A HPLC/GF AA procedure based on the use of C-18 columns is described for the quantitation of butyltin species in marine samples. When a mass spectrometer was used as detector (HPLC MS), evidence was obtained for the presence of other tin compounds in the samples. Extracts of samples were treated with CH₃MgBr and examined by using GC MS; the presence of Bu_nSnMe_4?n (n = 3–1, Bu = butyl) and Cy_nSnMe_4?n (n = 3,2, Cy = cyclohexyl) was confirmed in the derivatized seawater, bivalve flesh, and bivalve shell samples. Quantitative data are given for butyl- and cyclohexyl-tin species in seawater and the surface microlayer, and oyster flesh.