Simultaneous RP-LC Determination of Artesunate and Amodiaquine in Pharmaceutical Preparations

A simple, fast and precise reversed phase liquid chromatographic method was developed for the simultaneous determination of artesunate (AS) and amodiaquine (AD) in combined pharmaceutical dosage form. Chromatographic separation of the two drugs was performed on a BDS Hypersil C18, 100 mm × 4.6 mm, 3 μm particle size column as stationary phase with a mobile phase comprising of phosphate buffer (pH 3.0 with orthophosphoric acid) and acetonitrile in the proportion of 50:40 (v/v), at a flow rate of 0.8 mL min^?1 and UV detection at wavelength 210 nm for AS and 300 nm for AD using photo diode array detection. The proposed method was validated for specificity, accuracy, linearity, range, precision and was successfully applied to the simultaneous determination of AS and AD in the combined fixed dosage form without any excipient’s interference.     

Terahertz properties of fluorinated liquid crystals

In this article, optical properties of four fluoro-substituted 4-propyl-4′-[(4-ethylphenyl)ethynyl] biphenyls and liquid crystal mixture A are presented in the terahertz (THz) range. Birefringence, refractive indices and absorption coefficients for ordinary and extraordinary ray of liquid crystals are described in the range of 0.3 to 3.0 THz. It shows that the measured parameters are dependent on the number and placement of fluorine atoms in the molecules. Measurements have been performed using time-pulsed spectroscopy.     

Simultaneous Determination of Tetrahydropalmatine, Magnolol, Emodin and Chrysophanol in Chinese Herbal Preparation by RP-HPLC-PDA

A reversed phase high performance liquid chromatography coupled with photo-diode array (RP-HPLC-PDA) detection method was proposed for simultaneous determination of tetrahydropalmatine, magnolol, emodin and chrysophanol in a Chinese herbal preparation (Dan’an mixture). The separation was performed on a Diamonsil? C₁₈ column (250 × 4.6 mm, 5 μm) with methanol and 0.1% phosphoric acid (88:12, v/v) as the mobile phase at the flow-rate of 0.8 mL min^?1. Two detection wavelengths were utilized for the quantitative analysis (209 nm for tetrahydropalmatine and magnolol, and 220 nm for emodin and chrysophanol, respectively). A good linear regression relationship (r ≥ 0.9996) between peak-areas and concentrations was obtained over the range of 0.25–50 μg mL^?1 for all the analytes. The spike recoveries, measured at three concentration levels, varied from 90.13 to 102.11%. The method was successfully applied to determine the contents of the four compounds in Dan’an mixture.     

Simultaneous LC Estimation of Glimepiride and Metformin in Glimepiride Immediate Release and Metformin Sustained Release Tablets

In the present study, a simple, rapid and precise liquid chromatographic method was developed and validated for the simultaneous determination of glimepiride and metformin in sustained release formulation. The separation was achieved on a Nucleosil 100-5SA column, 250 × 4.6 mm i.d., 5 μm using a mobile phase composed of 1.7 % ammonium dihydrogen phosphate buffer pH 3.0: acetonitrile (70:30 v/v).The instrumental settings were a flow rate of 1.0 ml min^?1 and a detector wavelength of 230 nm. The retention time of glimepiride and metformin were 5.1 and 11.3, respectively. The developed method was validated in terms of specificity, sensitivity, linearity, range, accuracy, precision and ruggedness. The proposed method was successfully applied to the sustained release pharmaceutical dosage form for the simultaneous determination of glimepiride and metformin without any interference by excipients.     

Stability-Indicating UPLC Method for the Determination of Bisoprolol Fumarate and Hydrochlorothiazide: Application to Dosage Forms and Biological Sample

A sensitive, selective and accurate ultra performance liquid chromatographic method has been developed and validated for the simultaneous determination of bisoprolol fumarate and hydrochlorothiazide in their combined dosage forms and as well as in spiked human urine samples. The separation was achieved on an Acquity UPLC BEH C18 1.7 μm (2.1 × 50 mm) column, at 40 °C with mobile phase consisting of acetonitrile:phosphate buffer (20 mM) at pH 3.0 with a gradient elution at 225 nm. Bisoprolol fumarate and hydrochlorothiazide were well separated in <1.5 min with good resolution and without any tailing and interference of excipients. The method was fully validated according to ICH guidelines in terms of accuracy, precision, linearity and specificity. A linear response was observed over the concentration range 0.5–150 μg mL^?1 for hydrochlorothiazide and 0.5–250 μg mL^?1 for bisoprolol fumarate. Limit of detection and limit of quantitation for hydrochlorothiazide were calculated as 0.01 and 0.03 μg mL^?1, respectively, and for bisoprolol fumarate were 0.07 and 0.21 μg mL^?1, respectively. Moreover, bisoprolol fumarate and hydrochlorothiazide were subjected to degradation conditions such as hydrolytic, oxidative and thermal stress conditions to evaluate the ability of the proposed method for the separation of bisoprolol fumarate and hydrochlorothiazide from their degradation compounds.     

An LC Ion-Pairing Method for the Determination of Fe(II) in Ferrous Bisglycinate Pharmaceutical Formulation

A reversed-phase ion-pairing liquid chromatographic method was developed and validated for the assay of Fe(II) in ferrous bisglycinate (Fe-bis-gly) capsules using 4-(2-pyridylazo) resorcinol reagent. The analysis was carried out using a Gemini RP-18 (150 mm × 4.6 mm I.D., particle size 5 μm) analytical column; the mobile phase consisted of a mixture of acetonitrile–water (28:72 v/v) containing 1 mM tetrabutylammonium hydrogensulfate and 1% phosphate buffer (pH 8.0). The flow rate was 1.0 mL min⁻¹ and the detection was achieved with a photodiode array (PDA) detector at 706 nm. The specificity of the method was proved using stress conditions and evaluated using a PDA detector. The data validation showed that the method is specific, fast, accurate, and reproducible for the determination of Fe-bis-gly in dosage form. The response was linear over a range of 1.0–2.6 μg mL⁻¹ (r = 0.9999). The accuracy of the method ranged from 98.02 to 102.75%. The RSD values for intra- and inter-day precision studies were below 1.3 and 1.1%, respectively. There was no interference of the excipients on the determination of the active pharmaceutical ingredient.

     

Simultaneous LC Estimation of Glimepiride and Metformin in Glimepiride Immediate Release and Metformin Sustained Release Tablets

In the present study, a simple, rapid and precise liquid chromatographic method was developed and validated for the simultaneous determination of glimepiride and metformin in sustained release formulation. The separation was achieved on a Nucleosil 100-5SA column, 250 × 4.6 mm i.d., 5 μm using a mobile phase composed of 1.7 % ammonium dihydrogen phosphate buffer pH 3.0: acetonitrile (70:30 v/v).The instrumental settings were a flow rate of 1.0 ml min⁻¹ and a detector wavelength of 230 nm. The retention time of glimepiride and metformin were 5.1 and 11.3, respectively. The developed method was validated in terms of specificity, sensitivity, linearity, range, accuracy, precision and ruggedness. The proposed method was successfully applied to the sustained release pharmaceutical dosage form for the simultaneous determination of glimepiride and metformin without any interference by excipients.

     

Pre-Column Derivatization HPLC Procedure for the Quantitation of Aluminium Chlorohydrate in Antiperspirant Creams Using Quercetin as Chromogenic Reagent

This article describes the development and validation of a selective high-performance liquid chromatography method that allows, after liquid–liquid extraction and pre-column derivatization reaction with quercetin, the quantification of aluminium chlorohydrate in antiperspirant creams. Chromatographic separation was achieved on an XTerra MS C18 analytical column (150 × 3.0 mm i.d., particle size 5 μm) using a mobile phase of acetonitrile:water (15:85, v/v) containing 0.08 % trifluoroacetic acid at a flow rate of 0.30 mL min^?1. Ultraviolet spectrophotometric detection at 415 nm was used. The assay was linear over a concentration range of 3.7–30.6 μg mL^?1 for aluminium with a limit of quantitation of 3.74 μg mL^?1. Quality control samples (4.4, 17.1 and 30.6 μg mL^?1) in five replicates from five different runs of analysis demonstrated intra-assay precision (% coefficient of variation <3.8 %), inter-assay precision (% coefficient of variation <5.4 %) and an overall accuracy (% recovery) between 96 and 101 %. The method was used to quantify aluminium in antiperspirant creams containing 11.0, 13.0 and 16.0 % (w/w) aluminium chlorohydrate, respectively.     

Microwave-Assisted Extraction of Melamine Residues from Pet Food and Analysis by Ion-Exchange LC–DAD

Melamine is attracting much attention because of its toxicity. In the work discussed in this paper, microwave-assisted extraction in combination with ion-exchange high-performance liquid chromatography with diode-array detection was used for analysis of melamine in pet food. Trichloroacetic acid–N,N-dimethylformamide 10:1 was the best extractant, because of the strong polarity of melamine. Separation was performed on a 250 mm × 4.6 mm i.d., 5-μm particle, cation-exchange column; isocratic elution with a mixture of ammonium formate solution (pH 3.0) and acetonitrile was complete within 10 min. UV absorbance DAD detection was performed at 240 nm. Response was a linear function of melamine concentrations from 0.1 to 50 μg mL^?1, with a detection limit of 1.0 mg kg^?1. Intra-day and inter-day precision, as RSD, was <3% and the recovery of the assay was in the range 95.4–106.8%. In analysis of spiked pet food, the new method yielded satisfactory results. Because of its simplicity and accuracy this straightforward method is particularly suitable for routine melamine analysis.     

LC Determination of Clindamycin Phosphate from Chitosan Microspheres

A simple, rapid and precise reverse phase LC method was adopted, modified and validated for the determination of clindamycin phosphate from chitosan microspheres prepared by spray drying method. Separation was performed using ACE5 C18 reversed phase column (150 mm × 4.6 mm, 5 μm) with acetonitrile:phosphate buffer at pH 2.5 (25:75 v/v) as mobile phase. The limit of detection was 46.43 × 10^?3 μg mL^?1, with UV detection at 210 nm. No interference from chitosan and other excipients was observed. Therefore an incorporation efficiency of microspheres could be determined accurately and specifically.     

Rapid LC-UV Analysis of Iohexol in Canine Plasma for Glomerular Filtration Rate Determination

A rapid high-performance liquid chromatography UV method and a simple sample preparation for analyzing iohexol in canine plasma, for evaluating glomerular filtration rate (GFR) and intestinal permeability, were developed and validated. Trifluoroacetic acid (TFA) was used for protein precipitation and iohexol extraction from plasma, followed by vortex mixing and centrifugation. As an internal standard, 4-aminobenzoic acid (para-aminobenzoic acid, PABA) was added. The supernatant (5 μL) was injected into a Zorbax SB-C18 LC column maintained at 50 °C. The mobile phase of the LC method was a water–methanol gradient at pH 3.0 adjusted with TFA. Fast LC measurement was achieved by using a rapid-resolution LC technique. Total run time was 13 min, and UV wavelength was set at 246 nm. Precision of the method was 0.2–9.0%, depending on the iohexol concentration in plasma. Recovery of iohexol from plasma was over 90%, and recovery of the internal standard 99.1 ± 1.4%. The calibration curve was linear (r = 0.9997) over iohexol concentrations of 2.5–150 μg mL^?1 (n = 5). This method is fast, simple, reliable and applicable in clinical settings.     

Development and Validation of an HPLC–UV Method for the Determination of Insulin in Rat Plasma: Application to Pharmacokinetic Study

A simple, sensitive high performance liquid chromatographic method with UV detection was developed and validated for determination of insulin in rat plasma, using methyl paraben as an internal standard. Insulin was extracted from plasma by a liquid–liquid extraction with a mixture of dichloromethane and n-hexane (1:1, v/v) followed by an acidic back extraction. Chromatographic separation was achieved isocratically with a Phenomenex® C₁₈ analytical column (150 × 4.6 mm ID, 5 μm) at ambient room temperature. The calibration curves were linear within a concentration range of 0.7–8.4 μg mL^?1 (r ² = 0.9994). The inter-day and intra-day accuracy and precision were ≤3.33 and ≤5.55%. The limit of detection (LOD) and limit of quantification (LOQ) were 0.35 and 0.7 μg mL^?1. The average recovery was 87.86% for insulin and 83.52% for methyl paraben. Insulin containing plasma samples were stable at ?20 °C for 7 days. Validated HPLC method was successfully applied to a pharmacokinetic study of insulin in streptozotocin induced diabetic rats.     

Assay of Kanamycin A by HPLC with Direct UV Detection

The development of a simple reversed phase ion pair liquid chromatographic method for the assay of kanamycin A has been described. Because of the lack of a UV chromophore in the structure of kanamycin A, borate complexation was used to allow direct UV detection at 205 nm. Three columns were evaluated in this study: Zorbax Extend C18 (4.6 mm × 250 mm; 5 μm), XBridge C18 (4.6 mm × 250 mm; 5 μm) and apHera C18 (4.6 mm × 250 mm; 5 μm). The mobile phase was a mixture of 0.1 M disodium tetraborate (pH 9.0) and water (20:80, v/v) supplemented with 0.5 g L^?1 sodium octanesulphonate. Final chromatographic conditions were achieved on the XBridge column at 50 °C. The method was validated according to ICH guidelines and applied to a commercially available sample. It is much faster and more specific than the current microbiological assay prescribed in the European Pharmacopoeia. No expensive equipment is necessary to perform this assay making it a viable replacement.     

Development and Validation of an Accurate LC Method for the Quantitative Determination of Voriconazole in a New Emulsion Formulation

A simple, sensitive and accurate liquid chromatographic method with UV detection was developed and validated to determine voriconazole in a new emulsion formulation. Chromatographic separation was achieved on a Diamonsil C₁₈ column (250 × 4.6 mm I.D., 5 μm) using a mobile phase consisting of acetonitrile-water-acetic acid (40:60:0.25, v/v/v) at a flow rate of 1.0 mL min^?1. The UV detection wavelength was set at 256 nm. The linear calibration curves were obtained in the concentration range of 1.00–100 μg mL^?1 with the limit of quantification of 1.00 μg mL^?1. The within- and between-run precisions in terms of percentage relative standard deviation were lower than 7.4 and 7.1%, respectively. The accuracy in terms of percentage relative error ranged from ?1.5 to 1.4%. This validated method was successfully applied to the determination of the content of voriconazole in a new emulsion formulation.     

Assay of Kanamycin A by HPLC with Direct UV Detection

The development of a simple reversed phase ion pair liquid chromatographic method for the assay of kanamycin A has been described. Because of the lack of a UV chromophore in the structure of kanamycin A, borate complexation was used to allow direct UV detection at 205 nm. Three columns were evaluated in this study: Zorbax Extend C18 (4.6 mm × 250 mm; 5 μm), XBridge C18 (4.6 mm × 250 mm; 5 μm) and apHera C18 (4.6 mm × 250 mm; 5 μm). The mobile phase was a mixture of 0.1 M disodium tetraborate (pH 9.0) and water (20:80, v/v) supplemented with 0.5 g L⁻¹ sodium octanesulphonate. Final chromatographic conditions were achieved on the XBridge column at 50 °C. The method was validated according to ICH guidelines and applied to a commercially available sample. It is much faster and more specific than the current microbiological assay prescribed in the European Pharmacopoeia. No expensive equipment is necessary to perform this assay making it a viable replacement.

     

LC–MS Determination of Gabapentin from Human Plasma

Gabapentin is an anticonvulsant drug used for the treatment of epilepsy. It is not bound to plasma protein and is not metabolized. A high performance liquid chromatography–mass spectrometric micro method is described in this report for its determination from human plasma. Chromatography was performed on a 50 × 4.6 mm, 4 μm nitrile column and the parent ion detected in the positive ionization mode on single quadrupole analyzer (Q1MI) with atmospheric pressure ionization source. Extraction was carried out on C₁₈, 100 mg/3cc cartridge using 10 μL sample volume. The mean extraction recovery was 97% and within batch and between batch coefficients of variation were <9%. Lack of interference from endogenous substances helped in achieving a highly sensitive method without the need for monitoring fragment ions. The lowest concentration injected on column for calibration curve was 195 pg (range 0.5–64 ng). The method was applied for analysis of samples from a cross-over bio-equivalence study comparing two formulations.

     

LC–MS Determination of Gabapentin from Human Plasma

Gabapentin is an anticonvulsant drug used for the treatment of epilepsy. It is not bound to plasma protein and is not metabolized. A high performance liquid chromatography–mass spectrometric micro method is described in this report for its determination from human plasma. Chromatography was performed on a 50 × 4.6 mm, 4 μm nitrile column and the parent ion detected in the positive ionization mode on single quadrupole analyzer (Q1MI) with atmospheric pressure ionization source. Extraction was carried out on C₁₈, 100 mg/3cc cartridge using 10 μL sample volume. The mean extraction recovery was 97% and within batch and between batch coefficients of variation were <9%. Lack of interference from endogenous substances helped in achieving a highly sensitive method without the need for monitoring fragment ions. The lowest concentration injected on column for calibration curve was 195 pg (range 0.5–64 ng). The method was applied for analysis of samples from a cross-over bio-equivalence study comparing two formulations.     

A Stability-Indicating Assay of Brimonidine Tartrate Ophthalmic Solution and Stress Testing Using HILIC

A stability-indicating hydrophilic interaction liquid chromatography (HILIC) method has been developed and validated for the quantitative determination of Brimonidine tartrate (BT) formulated as an ophthalmic solution. Isocratic separation was achieved using an acetonitrile-buffer mixture (92:8, v/v) at pH 7.1 on an unmodified silica column (250 × 4.6 mm, 5 μm). The drug was subjected to oxidative, hydrolytic, photolytic and thermal stress conditions and complete separation was achieved for the parent compound and degradation products. The influence of acetonitrile, pH and ionic strength of the buffer was studied. Linearity range and recoveries for BT were 100–400 μg mL^?1 and 100.12%, respectively. The method was validated for BT and indicated that the method was sufficiently sensitive with a limit of detection at 0.005 μg mL^?1 and a limit of quantitation at 0.02 μg mL^?1, respectively.     

Establishment of an HPLC method for testing acetylcholinesterase inhibitory activity and compared with traditional spectrophotometry

Acetylcholinesterase (AChE) inhibitory activity test is one of the important areas of natural product research, which is traditionally tested by spectrophotometry. In this work, a simple high-performance liquid chromatography (HPLC) method was developed for testing AChE inhibitory activity in several natural products. Using galanthamine hydrobromide and huperzine A as positive controls, it was found that the optimal conditions for sample preparation are 7.0–8.0 for pH, 30 min for reaction time, 37 °C for reaction temperature, 1.1 × 10^?3 U mL^?1 for concentration of AChE, and 0.625 mM for acetylthiocholine iodide in the final reaction mixture with total volume 2 mL. The Diamonsil C₁₈ column was used and the chromatographic conditions were mobile phase methanol–water–triethylamine (40:60:0.05, v/v/v), flow rate 1.0 mL min^?1, injection volume 5 μL, run time 5 min, column temperature 37 °C, and wavelength 405 nm. The AChE inhibitory activity of anthocyanin with concentration of 10, 20, and 50 μg mL^?1, blueberry extract and purple potato extract with concentration of 20, 100, and 500 μg mL^?1 were further determined under this conditions. The results showed the inhibition ratios obtained from spectrophotometry were nearly adjacent and approximately lower than 10% and the values of absorbance mostly exceed 0.8, but the absorbance should ensure in the range of 0.15–0.8 to guarantee smaller relative error for spectrophotometry. While the inhibition ratios gained from HPLC method were higher than 20% and had greater difference, they had better comparability to the different concentrations of anthocyanin and its extracts. It is revealed that the HPLC method can be used for the evaluation of the AChE inhibitory activity in natural product extracts with deep color or not.     

Determination of 2,6-di-tert-butylphenol in Irganox 1425

An ion suppression reversed phase high performance liquid chromatography method has been developed for the determination of 2,6-di-tert-butylphenol in Irganox 1425. Separation was carried out on a Welch Material Ultimate XB-C₁₈ column (5 μm, 150 mm × 4.6 mm), using methanol–water (0.1 % HClO₄) (75/25, v/v) mixture as the mobile phase in an isocratic elution. Detection was performed by UV absorption at a wavelength of 275 nm, and the limit of detection was 0.2 μg/mL. The method presented excellent repeatability and precision with both intra- and inter-day relative standard deviations (RSDs) of less than 2.0 %, as well as satisfactory accuracy with the recovery of 98.7–102.3 %, and therefore can be applied for the quality control analysis of Irganox 1425 industrial product.