Quantitative Analysis of Pentalgin ICN Tablets by Gradient and Isocratic High-Performance Liquid Chromatography

Two procedures were proposed for determining analgin (dipyrone), paracetamol, caffeine, phenobarbital, and codeine in the multicomponent drug Pentalgin ICN with the use of HPLC in gradient and isocratic modes. In the gradient version, columns packed with Nucleosil 100 C18 or Nova-Pak C18 adsorbents were used; at the initial stage, an acetonitrile-water mixture was used as an eluant, and a 0.025 M KH₂PO₄ solution was introduced as a mobile phase constituent after the emergence of all peaks other than that of codeine or immediately after the emergence of the analgin peak. In the isocratic version, a Nova-Pak CN HP column was used; a 1% KH₂PO₄ solution containing 5 vol % acetonitrile was an eluant. The detector wavelength was 212 nm. The separation time was shorter than 10 min (mobile-phase flow rate of 1 mL/min). Model solutions containing all active components and additives of the tablets were analyzed, and the performance characteristics of both procedures were calculated. The procedures ensure reliable analytical results; however, the isocratic version is technically simpler and more preferable for product control in commercial manufacture.     

Quantitative analysis of Pentalgin tablets by gradient and isocratic high-performance liquid chromatography

The retention of paracetamol, propyphenazone, caffeine, phenobarbital, and codeine phosphate, which are the components of the new medicine Pentalgin, was studied by reversed-phase high-performance liquid chromatography on a column (150 × 3.9 mm) filled with the Symmetry C18 sorbent (5.0 μm) in the gradient elution mode and on a column (150 × 3.9 mm) filled with the Nova-Pak CN HP sorbent (4.0 μm) as a function of the profile and composition of the gradient and as a function of the concentrations of acetonitrile and KH₂PO₄ and the pH of the mobile phase, respectively, with detection at 212 nm. The optimum composition of the mobile phase was selected. The time of separation was 16 and 11 min for the gradient and isocratic elution modes, respectively. The procedures were used for the analysis of a preproduction sample of the tablets. The procedures provide accurate and reproducible results of analysis; however, the isocratic version is preferable for mass production control as a technically simpler technique.     

Determination of Amphotericin-B in Human Plasma by Reversed-Phase High Performance Liquid Chromatography Using a Short Octyl Column

Abstract

Amphotericin-B is a polyene antifungal antibiotic used for the treatment of severe systemic fungal infections. For effective treatment of urinary fungaria and the prevention of significant adverse-effects, monitoring the concentration of Amphotericin-B in biological samples of humans (ingesting the drug) is required. In this experiment, Amphotericin-B was isolated from plasma endogenous substances by adding 200 μL of acetonitrile in 800 μL of plasma. This mixture was vortex mixed, 20 mg of zinc sulfate and 10 mg of monobasic potassium phosphate was added to the mixture. This mixture was again vortex mixed and followed by centrifugation. The supernatant was filtered through a 0.45 μm membrane and a 100 μL aliquot of this solution was injected onto the chromatographic system. A short column of 60 mm × 4.6 mm packed with 3 μm octyl particles was used with an isocratic elution of 50/50, acetonitrile/0.01M KH₂PO₄ (v/v). The pH of the mobile phase mixture was adjusted to 3.5 with H₃PO₄. The intact drug molecule (parent drug) was monitored by a W-visible detector at 410 nm and 0.10-0.005 A.U.F.S. The limits of detection of the method were 0.03 μg/mL for 100 μl injection volume at signal-to-noise ratio of 3.     

Analysis of Ribonucleotides by Reverse-Phase HPLC Using Ion Pairing on Radially Compressed Or Stainless Steel Columns

Abstract

A method is described for the analysis of 16 major nucleotides including NAD, IDP, GDP, cAMP, cGMP and succinyl AMP by reverse phase HPLC. The use of 65mM KH₂PO₄, at pH 3.2, low concentrations of the ion pairing reagent tetrabutylammonium phosphate and acetonitrile allowed the simultaneous separation of these nucleotides by isocratic or gradient elution in 18 and 28 minutes respectively. Stainless steel and radially compressed columns were compared and a similar separation profile was obtained. The latter columns increased retention and improved the efficiency of separation.     

Analysis of Invertebrate Neuropeptides by RP-HPLC

Abstract

The application of reverse phase-high performance liquid chromatography to the analysis of four synthetic invertebrate neuropeptides is described. Proctolin (Arg-Try-Leu-Pro-Thr), locust adipokinetic hormone (p-Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH₂), crustacean erythrophore concentrating hormone (p-Glu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH₂) and mulluscan cardioexcitatory neuropeptide (Phe-Met-Arg-Phe-NH₂) were analyzed on several different reverse phase columns by means of gradient elution with 0.01M KH₂ PO₄, 0.1% H₃PO₄, 0.25N triethylammonium phosphate (TEAP), pH 2.20, or 0.1% trifluoroacetic acid (TFA) versus acetonitrile. Column effluents were monitored at both 254 and 195 nm except in the case of TFA where 254 and 210 nm were monitored. At the lower wavelength computerized background correction was sometimes necessary to correct excessive baseline drift during the course of the gradient run. Best results were obtained on the Supelcosil LC-18DB column with a concave gradient of 90° 40%B over 1 hr at 1.1 ml/min where B[dbnd]0.25N TEAP, pH 2.20, A[dbnd]acetonitrile. With this system less than 5 ng of peptide was detectable. The use of the volatile TFA buffer permitted recovery of peptides from the column effluent by lyophilization.     

Application of gradient high-performance liquid chromatography to the analysis of some multicomponent pharmaceutical preparations

Procedures were developed for determining streptocide and norsulfazole in the Ingalipt aerosol; dexpanthenol and nipagin in the Dekspantenol aerosol; and oxymetazoline and benzalkonium chloride in a nasal spray by reversed-phase high-performance liquid chromatography (HPLC) in the gradient elution mode using the CH₃CN-0.025 M aqueous solution mixture of KH₂PO₄ (pH 3.0) and UV detection. The developed procedures are comparable in accuracy and precision to chromatographic procedures recommended by regulatory documents; however, they ensure the simultaneous determination of all components of the pharmaceutical preparations and are more rapid and less laborious.     

Separation of Methylated Purines by High Pressure Liquid Chromatography

Abstract

This paper reports a method for the separation and measurement of methylated purines of interest to carcinogenesis studies by high-pressure liquid chromatography (HPLC) following their column chromatographic isolation from collected urine samples. HPLC was evaluated on three different cation-exchange columns, with optimum conditions obtained on a Partisil 10-SCX column employing isocratic elution with 0.25M (NH₄)H₂PO₄ at pH 4.0. This column was also found to be useful for the separation of mono-methylguanine isomers. Application is shown to the analysis of rat urine following animal treatment with methyl methanesulfonate.     

Chromatographic methods development,validation and degradation characterization of the antithyroid drug Carbimazole

The current paper reports the development and validation of stability‐indicating HPLC and HPTLC methods for the separation and quantification of main impurity and degradation product of Carbimazole. The structures of the degradation products formed under stress degradation conditions, including hydrolytic and oxidative, photolytic and thermal conditions, were characterized and confirmed by MS and IR analyses. Based on the characterization data, the obtained degradation product from hydrolytic conditions was found to be methimazole—impurity A of Carbimazole as reported by the British Pharmacopeia and the European Pharmacopeia. A stability‐indicating HPLC method was carried out using a Zorbax Eclipse Plus CN column (150 × 4.6 mm i.d, 5 μm particle size) and a mobile phase composed of acetonitrile–0.05 m KH₂PO₄ (20: 80, v/v) in isocratic elution, at a flow rate of 1 mL/min. The method was proved to be sensitive for the determination down to 0.5% of Carbimazole impurity A. Additionally, a stability‐indicating chromatographic HPTLC method was achieved using cyclohexane–ethanol (9:1, v/v) as a developing system on HPTLC plates F₂₅₄ with UV detection at 225 nm. The proposed HPLC and HPTLC methods were successfully applied to Carbimazole^® tablets with mean percentage recoveries of 100.12 and 99.73%, respectively.     

Simultaneous Determination of Abacavir,Efavirenz and Valganciclovir in Human Serum Samples by Isocratic HPLC-DAD Detection

A rapid, sensitive, and specific reverse phase high performance liquid chromatography with diode array detection procedure for the simultaneous determination of abacavir, efavirenz and valganciclovir in spiked human serum is described. Separation was performed on a 5 μm Waters Spherisorb column (250 × 4.6 mm ID) with acetonitrile: methanol:KH₂PO₄ (at pH 5.00) (40:20:40 v/v/v) isocratic elution at a flow rate of 1.0 mL min⁻¹. Calibration curves were constructed in the range of 50–30,000 ng mL⁻¹ for abacavir and efavirenz, and 10–30,000 ngmL⁻¹ for valganciclovir in serum samples. The limit of detection and limit of quantification concentrations of the HPLC method were 3.80 and 12.68 ng mL⁻¹ for abacavir, 2.61 and 8.69 ng mL⁻¹ for efavirenz, 1.30 and 4.32 ng mL⁻¹ for valganciclovir. The method has been applied, without any interference from excipients or endogenous substances, for the simultaneous determination of these three compounds in human serum.

     

Influence of isomerism on the chromatographic behaviour of porphyrins

Summary The chromatographic behaviour of some porphyrins and their complexes with zinc has been studied by HPLC on 150×3.9 mm and 300×3.9 mm columns packed with Nova-Pak C₁₈ and μ-Bondapak C₁₈, respectively, and on a microcolumn (64×2 mm) packed with Nucleosil C₁₈. The effect of the nature and the arrangement of side substituents in the porphyrin molecules on retention is considered. It is demonstrated that HPLC can be used for the separation ofcis-andtrans-isomers (atropisomers) of the zinc complex of 5,15-di(phenyl-2-CH₃O)-3,7,13,17-tetramethyl-2,8,12,18-tetrabutylporphyrin and other porphyrins with a similar structure. The efficiency of separation has been compared on different columns.     

Optimization of chromatography to overcome matrix effect for reliable estimation of four small molecular drugs from biological fluids using LC–MS/MS

The article describes a systematic study to overcome the matrix effect during chromatographic analysis of gemfibrozil, rivastigmine, telmisartan and tacrolimus from biological fluids using LC–ESI–MS/MS. All four methods were thoroughly developed by the appropriate choice of analytical column, elution mode and pH of mobile phase for improved chromatography and overall method performance. Matrix effect was assessed by post-column analyte infusion, slope of calibration line approach and post-extraction spiking. The best chromatographic conditions established were: Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) column with 5.0 mm ammonium acetate, pH 6.0–methanol as the mobile phase under gradient program for gemfibrozil; Luna CN (50 × 2.0 mm, 3 μm) column with a mobile phase consisting of acetonitrile–10 mm ammonium acetate, pH 7.0 (90:10, v/v) for rivastigmine; Inertsustain C₁₈ (100 × 2.0 mm, 5 μm) column using methanol–2.0 mm ammonium formate, pH 5.5 (80: 20, v/v) as the mobile phase for isocratic elution of telmisartan; and Acquity BEH C₁₈ (50 × 2.1 mm, 1.7 μm) with methanol–10 mm ammonium acetate, pH 6.0 (95:5, v/v) as mobile phase for tacrolimus. The methods were thoroughly validated as per European Medicines Agency and US Food and Drug Administration guidance and were successfully applied for pharmacokinetic studies in healthy subjects.     

A Chemometrical Approach to Optimization and Validation of an HPLC Assay for Rizatriptan and its Impurities in Tablets

Abstract

An isocratic reversed‐phase high‐performance liquid chromatographic method was developed and validated for the analysis of a novel antimigraine drug, rizatriptan benzoate, in a dosage form along with its two impurities, L‐749.019 and L‐783.540. The method used a C₁₈ XTerra? (150×3.9 mm), 5 µm column. The mobile phase consisted of a mixture of methanol, TEA (1%) and 10 mM KH₂PO₄ (5:9.5:85.5 v/v) at a flow rate of 1.2 ml min^?1 (pH of the water phase was adjusted to 5.5 with 85% orthophosphoric acid). Column temperature was 20°C and the detection was performed at 225 nm. The central composite design technique and the response surface method were used in the robustness test considerations. The method was applied satisfactorily to the analysis of commercial rizatriptan formulation.     

Versatile online SPE–HPLC method for the analysis of Irinotecan and its clinically relevant metabolites in biomaterials

Monitoring levels of Irinotecan and its metabolites during cancer therapy could help link broad interpatient variations in antitumor activity and toxicity to the patient's metabolic status. We have developed and validated a versatile and highly sensitive method for the simultaneous determination of Irinotecan and its clinically relevant metabolites 7‐ethyl‐10‐hydroxy‐camptothecin (SN‐38) and SN‐38 glucuronide. Sample clean‐up involves precipitation by acetone/methanol/0.5 M trichloroacetic acid at 4:4:2 v/v followed by extraction of the metabolites on an SPE column by 20% methanol in 25 mM KH₂PO₄ pH 2.9. Online transfer to an analytical μBondapak C18 column, elution with 24% acetonitrile (ACN) in 0.1 M KH₂PO₄ pH 2.9 and fluorescence detection with excitation at 375 nm and emission at 430 nm for SN‐38 glucuronide and Irinotecan or 540 nm for SN‐38 results in high sensitivity (1–2 pg) and short (～10 min) run times. The method was used to determine the degree of SN‐38 glucuronidation in mice after Irinotecan administration and in cultured cancer cells exposed to SN‐38. The method may be used to better understand Irinotecan metabolism, personalize therapy, and develop Irinotecan‐based tumor targeting therapies.     

Axial temperature gradient and mobile phase gradient in microcolumn high-performance liquid chromatography

The effect of axial temperature gradient (ATG) along a microcolumn on the separation performance at both isocratic and gradient elution mode was investigated. A thermostat system was designed to form an ATG along the packed column. Polycyclic aromatic hydrocarbons (PAHs) were separated on a 0.53 mm  × 150 mm i.d. 5 μm C₁₈ microcolumn, with water and acetonitrile as mobile phase. The separation results obtained at mobile phase gradient (MPG) and ATG in microcolumn HPLC were compared with the results performed at ambient conditions. Extrapolated curves of peak width at half height (wh)versus lnk showed that wh is narrower at the same retention time when ATG was applied in addition to MPG. The column efficiency was enhanced 20-30% and the resolution was slightly reduced because of reduction of selectivity at elevated temperature at ATG condition. The RSD of retention time in ATG mode was less than 2.5%.     

Determination of Organic Acids in Red Wine and Must on Only One RP-LC-Column Directly After Sample Dilution and Filtration

A new method for simultaneous determination of organic acids in red wine and must by liquid chromatography was studied. The determination of organic acids in wines can be achieved in less than 13 min, preceded only by a simple sample dilution and filtration step. With this method, the chromatographic separation of eight organic acids and interfering peaks present in red wine, required only one reversed phase column (Waters Atlantis dC18 column, 4.6 × 150 mm ID, 5 μm). As mobile phase, isocratic acetonitrile–0.01 mol L^?1 KH₂PO₄ at pH 2.7 5:95 (v/v) at a flow rate of 0.8 mL min^?1 was used. Detection wavelength was set at 210 nm except for ascorbic acid which was detected at 243 nm. Application to red wine and must confirmed good repeatability and showed a wide variation range for concentrations of organic acids.     

Determination of cysteinesulfinate, hypotaurine and taurine in physiological samples by reversed-phase high-performance liquid chromatography

Cysteinesulfinate, hypotaurine and taurine, which are key metabolites of cysteine, can be separated from each other and other closely eluting amino acids in biological samples by reversed-phase high-performance liquid chromatography on a Waters Nova-Pak C18 column. Samples were derivatized with o-phthalaldehyde-2-mercaptoethanol prior to injection. The elution system consisted of 100 mM potassium phosphate buffer, pH 7.0, with 3% (v/v) tetrahydrofuran with an initial isocratic phase at 1.2% acetonitrile and a gradient from 1.2 to 12.8% acetonitrile. This method is suitable for measurement of the production of metabolites from cysteine by isolated cells and for analysis of plasma and tissue extracts. Low levels of hypotaurine in rat tissues were easily measured with this method and are reported here for the first time.     

Robust design space development for HPLC analysis of five chemical components in Panax notoginseng saponins

The present work describes the systematic development of a robust, precise, and rapid reversed-phase liquid chromatography method for the simultaneous analysis of five chemical components in Panax notoginseng saponins (PNS) using quality by design (QbD). The method was developed in two main phases: screening and optimization. During the screening phase, the most suitable stationary phase, column temperature, and flow rate were identified, while the secondary influential parameters, such as the gradient slope, the initial concentration of acetonitrile, and the initial isocratic hold of the gradient elution system were fine-tuned in the later optimization phase. In this phase, a 17-run experiment was used to examine multifactorial effects of these parameters on the critical resolution, analysis time, and peak symmetry. The Monte Carlo simulation method was successfully used to build the chromatographic design space and the process capability index C_p was introduced to evaluate the robustness of the design space. At last, the verification experiment was performed within the working design space by the accuracy profile methodology and model was found to be accurate. A robust combination of separation conditions predicted in the design space was obtained with the gradient slope of 1.6% · min^?1, the initial concentration of acetonitrile of 20%, and the initial isocratic hold of 20 min, and the total analysis time was no more than 40 min. The results demonstrated that rapid separation of five components of PNS herbal extract could be achieved on the chromatographic column packed with narrow size particles at backpressures available in ordinary high performance liquid chromatography (HPLC) instruments.     

The Rapid Determination of Neopterine in Human Urine by Isocratic High-Performance Liquid Chromatography

Abstract

An isocratic high-performance liquid chromatography method is described for the determination of Neopterine eliminated in human urine, using a μ-Bondapak C₁₈ column (300 × 3.9 mm I.D.) and a strongly polar phosphate buffer (pH 6.2) for elution. This analysis requires only 15 minutes and allows very good reproduc-tibility of retention times. This method is well-suited for automation and routine clinical laboratory in order to quantify human urinary Neopterine in healthy subjects and in subjects with malignant disorders.     

Simple HPLC method for detection of trace ephedrine and pseudoephedrine in high-purity methamphetamine

A simple and sensitive HPLC technique was developed for the qualitative determination of ephedrine and pseudoephedrine (ephedrines), used as precursors of clandestine d‐methamphetamine hydrochloride of high purity. Good separation of ephedrines from bulk d‐methamphetamine was achieved, without any extraction or derivatization procedure on a CAPCELLPACK C₁₈ MGII (250 × 4.6 mm) column. The mobile phase consisted of 50 mM KH₂PO₄–acetonitrile (94:6 v/v %) using an isocratic pump system within 20 min for detecting two analytes. One run took about 50 min as it was necessary to wash out overloaded methamphetamine for column conditioning. The analytes were detected by UV absorbance measurement at 210 nm. A sample (20 mg) was simply dissolved in 1 mL of water, and a 50 μL aliquot of the solution was injected into the HPLC. The detection limits for ephedrine and pseudoephedrine in bulk d‐methamphetamine were as low as 3 ppm each. This analytical separation technique made it possible to detect ephedrine and/or pseudoephedrine in seven samples of high‐purity d‐methamphetamine hydrochloride seized in Japan. The presence of trace ephedrines in illicit methamphetamine may strongly indicate a synthetic route via ephedrine in methamphetamine profiling. This method is simple and sensitive, requiring only commonly available equipment, and should be useful for high‐purity methamphetamine profiling. Copyright © 2011 John Wiley & Sons, Ltd.     

Prediction of experimental parameters in combined isocratic and gradient elution reversed phase high performance liquid chromatography (RP-HPLC) using acetonitrile as organic modifier

The use of gradient elution in RP-HPLC is increasing. Aqueous buffers, such as phosphate at pH 1.7, with acetonitrile as the organic modifier are particularly advantageous as eluents with linear elution strength (LES). In the isocratic mode, under the experimental conditions used, fairly good linearity exists between logk' and x_B (x${}_{\text{B}}^{\text{º}}$ is the volume ratio of the organic modifier, i.e. acetonitrile) with a correlation coefficient of 0.9995. The relationship between isocratic and gradient elution parameters, such as x_B, x${}_{\text{B}}^{\text{º}}$ (starting composition in gradient elution), m (slope of the linear gradient), is transparent and the parameters of interest can be predicted fairly accurately using simple equations. The proposed system could be used in elaboration of a general retention index system for organic molecules in RP-HPLC.