High-pressure Liquid Chromatographic Method Research Articles

Development and Validation of an HPLC Method for the Simultaneous Analysis of Dextromethorphan HBr, Potassium Guaiacolsulfonate and Sodium Benzoate in Cough Mixture

A rapid, sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) method was developed for simultaneous determination of dextromethorphan hydrobromide (DXM), sodium benzoate (SB) and potassium guaiacolesulfonate (PGS) in cough mixture. The combined drug mixtures were separated through a Hichrom C8 (octyl), 25 cm x 4.6 mm ID, 5 μm column with a mobile phase composed of methanol and 10 mM phosphate buffer pH 3 (1:1), a flow rate 1ml/min, injection volume 10 μl and quantification by UV detection at λ max 219 nm. The method provided a short analysis time: PGS, tR = 3.07 min; SB, tR = 8.09 min and DXM, tR = 9.29 min. Validation was performed using raw materials and the pharmaceutical preparation which contains the compounds described above. The method was validated for linearity, precision and accuracy. Five factors were selected to be used in the robustness test (temperature of the column, volume of methanol in the mobile phase, pH of the aqueous phase in the mobile phase, flow rate and time of preparation) and the resolution between SB and DXM was measured for each factor. The statistical treatment (t-test with 4 degrees of freedom and at α = 0.01) did not show significant difference with regard to temperature of the column, pH of the aqueous phase in the mobile phase, flow rate and time of preparation showing that the method is robustic. With regard to percent organic modifier (methanol) a significant difference was observed indicating that care should be taken during the preparation of mobile phase proportions. Keywords: high pressure liquid chromatographic method, dextromethorphan HBr, sodium benzoate, potassium guaiacolsulfonate, cough mixtureEthiopian Pharmaceutical Journal Vol. 26 (1) 2008: pp. 39-48

Interference of labile glycated hemoglobin on HbA(1c) assay by a high pressure liquid chromatography method

HbA(1c) assay by high pressure liquid chromatography remains submitted to interferences, among which that of labile HbA(1c) in 1 to 2% of samples. We have evaluated the interference of labile HbA(1c) on HbA(1c) assay using Variant II analyzer (Biorad), by in vitro formation of labile glycated haemoglobin and by evaluation of two protocols of elimination of labile HbA(1c) (wash and incubation of red blood cells in saline solution, or incubation in the wash/dilution solution of the analyzer). Levels of labile HbA(1c) higher than 4.5 % lead to underestimation of HbA(1c). The different protocols tested proved efficient and were adapted to routine conditions. The fastest method is the incubation of red blood cells in the wash/dilution solution for at least two hours, or more if labile fraction is unusually high.

Quantification of plasma reduced glutathione, oxidized glutathione and plasma total glutathione in healthy cats.

Glutathione is an important intracellular tripeptide with multiple functions. Abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, no data regarding concentration of plasma glutathione are available for domestic cats. This study discusses the development of a rapid, simple high pressure liquid chromatography method for measurement of reduced glutathione (GSH), oxidized glutathione (GSSH) and total glutathione (GSHt) in plasma, for the purpose of establishing baseline data for future studies. The mean concentrations of GSH, GSSH and GSHt were 4.51+/-1 microM; 19.44+/-3.79 microM (expressed as GSH equivalent) and 23.59+/-3.89 microM, respectively. This is the first report of plasma glutathione concentrations in this species.

Separation and measurement of plant alkaloid enantiomers by RP‐HPLC analysis of their Fmoc‐Alanine analogs

Ammodendrine (1), anabasine (2) and coniine (3) can cause congenital malformations in livestock. They appear naturally in both enantiomeric forms, and can cause variable physiological responses. A method to measure the enantiomeric ratio of these natural toxins is needed. To develop a simple and economical method in order to determine the enantiomeric ratios of piperidine and pyrrolidine alkaloids in small samples of plant material. Mixtures of isolated or purified plant alkaloids were converted to their Fmoc-L-Ala-alkaloid analogues forming diastereomeric mixtures, which were then analysed by high pressure liquid chromatography (HPLC) with mass spectrometry (MS) and ultraviolet (UV) detection to determine enantiomeric ratios. The diastereomeric analogs for ammodendrine, anabasine and nornicotine could be separated and the enantiomeric ratios determined. The Fmoc-L-Ala-coniine analogue was not resolved under the HPLC conditions studied. The enantiomeric ratios of the selected plant alkaloids were measured and found to differ between both location within a species and location between species. A low-cost HPLC method to analyse the enantiomeric ratio of plant alkaloids containing primary or secondary amine nitrogens via conversion to their respective diastereomeric analogues has been developed.

Plasma assisted degradation of phenol solutions

Solutions of 2-chlorophenol, 4-chlorophenol and 2,6-dichlorophenol in bidistilled and water from the river Danube were treated in plasma reactor. In this reactor, based on coaxial dielectric barrier discharge at atmospheric pressure, plasma is formed over a thin layer of treated water. After one pass through the reactor, starting chlorophenols concentration of 20 mg/l was diminished up to 95%. Kinetics of the chlorophenols degradation was monitored by High Pressure Liquid Chromatography method (HPLC).

Synthesis of new carbon-11 labeled naphthalene-sulfonamides for PET imaging of human CCR8

Carbon-11 labeled naphthalene-sulfonamides, N-(4-( N-(4-[ 11C]methoxyphenyl)sulfamoyl)naphthalene-1-yl)benzamide ([ 11C] 5a), N-(4-( N-(4-[ 11C]methoxyphenyl)sulfamoyl)naphthalene-1-yl)-2-methylbenzamide ([ 11C] 5b), N-(4-( N-(4-[ 11C]methoxyphenyl)sulfamoyl)naphthalene-1-yl)-3-methylbenzamide ([ 11C] 5c), N-[ 11C]methyl- N-methyl-4-(4-benzamidonaphthalene-1-sulfonamido)piperidine-1-carboxamide ([ 11C] 9a) and N-[ 11C]methyl- N-methyl-4-(4-(2-methylbenzamido)naphthalene-1-sulfonamido)piperidine-1-carboxamide ([ 11C] 9b), have been synthesized as new potential positron emission tomography (PET) agents for imaging of human CCR8. The target tracers were prepared by either O-[ 11C]methylation or N-[ 11C]methylation of their corresponding precursors using [ 11C]CH 3OTf and isolated by either a simplified solid-phase extraction (SPE) purification procedure or a high pressure liquid chromatography (HPLC) method in 30–50% radiochemical yields decay corrected to end of bombardment (EOB), 20–25 min overall synthesis time, and 74–111 GBq/μmol specific activity at end of synthesis (EOS).

Gradient HPLC of antibiotics in urine, ground water, chicken muscle, hospital wastewater, and pharmaceutical samples using C‐18 and RP‐amide columns

A simple and highly sensitive high pressure liquid chromatographic (HPLC-UV) method has been developed for the determination of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, in mobile phase citrate buffer (0.001 M) of pH 4.5 prepared in water (X), methanol (Y), and ACN (Z) using gradient at a flow rate of 1.0 mL/min by direct UV absorbance detection at lambda = 280 nm. Separation of analytes was studied on the C-18 and RP-amide columns and best results were observed on the RP-amide column with LODs (3.3 x S/m) 0.89, 0.55, 0.67, and 1.41 ng/mL for ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid, respectively, and better RSD than the C-18 column. The recovery of Fluoroquinolones (FQs) in urine, ground water, hospital wastewater, and chicken muscle using this method is more than 90%. The method was successfully applied to the analysis of ofloxacin, lomefloxacin, cinoxacin, and nalidixic acid in urine, ground water, pharmaceutical dosage forms, hospital wastewater, and chicken muscle.

Analysis of hupehenine in the total alkaloids from Fritillaria hupehensis by HPLC-ELSD

A reverse-phase high pressure liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) has been developed for the quantitative analysis of hupehenine in the total alkaloids from Fritillaria hupehensis. Samples were analyzed on a reverse-phase column (Hypersil C-18) with a mobile phase of methanol:water:chloroform: triethylamine (85:15:1:0.6). The ELSD was set at the drift tube temperature of 68.3 degrees C and gas flow rate of 1.8 L/min. Hupehenine's retention time was 13.7 min with an asymmetry factor of 1.2. The validity of the method has been verified with linearity, limit of detection, accuracy and precision. The logarithmic linear curve was obtained from 8.936 to 134.04 microg/mL (r=0.9993). The detection limit (S/N>3) of hupehenine was 1.79 microg/mL on the column. Intra-day RSD was 1.42% and inter-day RSD was 2.26% (3 days within a week). The average recovery of hupehenine was 101.50%, and RSD was 1.62%.

The use of microcalorimetry and HPLC for the determination of degradation kinetics and thermodynamic parameters of Perindopril Erbumine in aqueous solutions

Perindopril Erbumine (PER) is one of the newly used angiotensin-converting enzyme inhibitors (ACE inhibitors) and is used for the treatment of patients with hypertension and symptomatic heart failure. It has two main degradation pathways, i.e. the degradation by hydrolysis and the degradation by cyclization. An isothermal heat conduction microcalorimetry (MC) and high pressure liquid chromatography (HPLC) were used for the characterization of aqueous solutions of PER and its stability properties. The rates of heat evolved during degradation of perindopril were measured by MC as a function of temperature and pH and from these data rate constant and change in enthalpy of the reactions were determined. With the HPLC method the concentration of perindopril and its degradation products were measured as a function of time in aqueous solutions of different pH that were stored at different temperatures. We demonstrated that reactions of degradation of perindopril at observed conditions follow the first order kinetics. The Arrhenius equation for each pH was determined. At pH 6.8 only one degradation pathway is present, i.e. the degradation by hydrolysis. Degradation constants for this pathway calculated from MC data are in good agreement with those obtained from HPLC. MC as a non-specific technique was shown to be useful in studies of PER when one reaction was present in the sample and also when more chemical and physical processes were simultaneously running.

A validated RP-HPLC method for simultaneous estimation of nebivolol and hydrochlorothiazide in tablets

A simple, selective, rapid, precise and economical reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of nebivolol and hydrochlorthiazide from pharmaceutical formulation. Phenomenex Gemini C18 (25 cm×4.6 mm i.d., 5 μ) column with a mobile phase consisting of acetonitrile: 50mM ammonium acetate (adjusted to pH 3.5 using orthophosphoric acid) (70:30 v/v) at a flow rate of 1.0 ml/min was used. Detection was carried out at 254 nm. Probenecid was used as an internal standard. The retention times of probenecid, nebivolol and hydrochlorthiazide were 13.05, 3.32 and 4.25 min, respectively. The developed method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantitation and solution stability. The proposed method can be used for the estimation of these drugs in combined dosage forms.

Preparation and testing of solutions for organ perfusion and preservation in transplantation

Histidine-tryptophan-ketoglutarate (HTK) solution is the most frequently used solution for the preservation of abdominal organs by leading transplant centers worldwide. The aim of the study was to investigate whether it is possible to prepare this kind of perfusion solution in the hospital pharmacy. In the hospital pharmacy setting we prepared HTK preservation solution and tested its quality, and in accordance with the obtained results recommended its shelf-life. A multicomponent HTK solution was prepared following standardized aseptic procedures for sterile products. Components of HTK solution were dissolved in water for injection under aseptic conditions and the obtained solution was sterilized by bacteriological membrane filtration (0.22 microm). The quality of the produced solution was examined using physical and physico-chemical methods and biological tests. Following pharmacopoeial regulations, we examined clarity, pH value, sterility and performed testing on pyrogens. The content of the amino acids histidine and triptophan was determined by using high pressure liquid chromatography (HPLC) method. The concentrations of chloride ions were investigated by retitration method. Atomic absorption spectrometry and ion-exchange chromatography with conductometric detection were used for determining the level of magnesium and calcium ions and the concentrations of sodium and potassium ions in HTK solution, respectively. It was shown that the quality of the prepared HTK solution complied with the regulation demands. Also, pH value of the solution as a function of storage temperature, remained in the physiological range (pH 7.4). According to the obtained results of physico-chemical and biological testing it can be concluded that in the hospital pharmacy setting with the applied technological procedure, we can produce HTK preservation solution of the required quality. Keeping of HTK preservation solution in a refrigerator at 4 degrees C warrants its shelf-life of six months.

Activated sludge-mediated biodegradation of dimethyl phthalate under fermentative conditions

The biodegradation of dimethyl phthalate (DMP) was investigated under fermentative conditions in this study. The nature of the intermediate compounds and the extent of mineralization were probed using high-pressure liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) methods. The fermentative bacteria were able to biodegrade the DMP under anaerobic conditions, with the biodegradation rate of 0.36 mg DMP/(L x h). The results demonstrated that the DMP degradation under fermentative conditions followed the modified Gompertz model with the correlation coefficient of 0.99. Monomethyl phthalate (MMP) and phthalic acid (PA) were detected as the intermediates of DMP biodegradation. During the experiment, MMP was rapidly produced and removed; however, PA accumulated as the biodegradation was slower throughout the course of the experiment. The COD(Cr) concentration decreased from 245.06 to 72.01 mg/L after the experimental operation of 20 d. The volume of methane produced was 3.65 ml over a period of 20 d and the amount of methane recovered corresponded to 40.2% of the stoichiometric value. The COD(Cr) variation and methane production showed that the DMP could not be completely mineralized under the fermentative conditions, which implied that the fermentative bacteria were not able to biodegrade DMP entirely.

Validation of a Simple and Rapid HPLC Method for Determination of Metronidazole in Dermatological Formulations

A rapid and simple method using an isocratic high-pressure liquid chromatography (HPLC) and UV detection for the determination of metronidazole in dermatological formulations is presented. Metronidazole samples were extracted with a solution composed of 60% methanol and 40% mobile phase by a procedure that can be completed in less than 10 min. Subsequent separation and quantification was accomplished in less than 20 min using reversed-phase HPLC with isocratic elution with 0.01% trifluoroacetic acid/acetonitrile (85:15%, vol/vol). Validation experiments confirmed the precision and accuracy of the method. When applied to a commercial metronidazole cream and gel formulation, recoveries of 100.4% for cream formulations and 102.3% for gel formulations were obtained. The method should facilitate studies of the formulation compatibility of metronidazole topical formulations with agents that may improve its clinical tolerability for treatment of rosacea.

Validação de metodologia para dosagem de porfirinas urinárias por cromatografia líquida de alta eficiência

Porphyrins are products that originate from the heme biosyntetic pathway. Enzymes that take part in this route can have their activity inhibited due to inherited/acquired or both factors resulting in increased serum heme precursor that will be eliminated in urine. Considering the importance of early detection of heme biosynthesis alterations, the purpose of this study was to establish a high pressure liquid chromatographic (HPLC) method with fluorescence detection, to detect five fractions of porphyrins: uroporphyrin (8-carboxyporphyrin), heptaporphyrin (7-carboxyporphyrin), hexaporphyrin (6-carboxyporphyrin), pentaporphyrin (5-carboxyporphyrin) and coproporphyrin I and III (4- carboxyporphyrin). Extraction methods with spectrophotometric detection are not sensitive enough for this purpose. The HPLC (Shimadzu Co., Kioto, Japan) was composed of two high-pressure pumps, auto-sampler and fluorescence detector (RF-535) with excitation at 400 nm and emission at 620 nm. The sample was eluted from a reversed-phase column with a linear gradient. The linearity of the method was from 8.0 to 120 µg/L for all fractions, proving its ablility to identify and quantify porphyrins with differents carboxylic groups for early diagnosis and follow-up of porphyrias.

Determination and Quantification of Phenytoin in Human Plasma by Liquid Chromatography with Electrospray Ionization Tandem Mass Spectrometry

A rapid and simple high pressure liquid chromatography method with mass spectrometry detection was developed and validated for the determination of phenytoin in human plasma. Metaxalone was used as internal standard. The sample preparation involves a rapid and simple procedure based on liquid‐liquid extraction. Analysis was performed in less than 3.0 minutes in isocratic mode on a reversed phase C18 column (5μ; 50 × 4.6 mm) using a mobile phase composed of acetonitrile‐buffer 2 mM ammonium acetate (80:20 v/v), pH of buffer adjusted to 3.4 using formic acid, at 0.4 mL min‐1 flow rate. The calibration curves were linear in the measured range between 101.2 ng mL-1 and 5060.0 ng mL-1. The validated lowest limit of quantification was 101.2 ng mL-1 for phenytoin. The mean relative recovery for drug and Internal standard was found to be 78.33% and 77.04%, respectively. The described method has the advantage of being rapid and easy and it could be applied in therapeutic monitoring of these drugs in human plasma.

Hemoglobin Ypsilanti

Hemoglobin (Hb) Ypsilanti is a rare high-oxygen-affinity hemoglobin. Like other high-oxygen-affinity hemoglobins, Hb Ypsilanti manifests as erythrocytosis. Because the migration of many high-oxygen-affinity variants on alkaline and acid gels does not differ from that of HbA, oxygen-hemoglobin dissociation studies are often used to document their presence. Hb Ypsilanti is a notable exception because its electrophoresis pattern on alkaline gel is highly characteristic, exemplifying the phenomenon of hybrid formation in variant hemoglobins. In the past few years, several laboratories have begun to use high-pressure liquid chromatography (HPLC) as a screen for hemoglobinopathies. We demonstrate the elution profile of Hb Ypsilanti on the 2 most widely used HPLC methods.

Simultaneous Determination of Diclofenac Sodium and Rabeprazole Sodium in Bulk and Pharmaceutical Dosage Form by LC

A simple, selective, rapid, precise and accurate reverse phase high pressure liquid chromatographic method has been developed for the simultaneous estimation of diclofenac sodium and rabeprazole sodium from pharmaceutical formulations. The method was developed using a HiQ SiL C18 (250 mm × 4.6 mm i.d.) column with a mobile phase consisting of methanol:water, (80:20 v/v), at a flow rate of 1.25 mL min−1. Detection was carried out at 284 nm. Indapamide was used as an internal standard. The developed method was validated for linearity, accuracy, precision, limit of detection and limit of quantitation. The proposed method can be used for the estimation of these drugs in combined dosage forms.

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Abstracts of Nippon Dojo-Hiryogaku Zasshi

Lutein and zeaxanthin in human milk independently and significantly differ among women from Japan, Mexico, and the United Kingdom

Abstract Human milk contains numerous dietary carotenoids, including lutein and zeaxanthin (LZ n=20 each) using a modified high-pressure liquid chromatography method. Mean L&Z concentrations in human milk differed among the 3 countries, with Japan having significantly more lutein than the United Kingdom ( P P r = 0.79, P

Comparative disposition of pharmacologic markers for cytochrome P‐450 mediated metabolism, glomerular filtration rate, and extracellular and total body fluid volume of Greyhound and Beagle dogs

The purpose of the study was to compare the disposition of pharmacologic markers for cytochrome P-450 (CYP) metabolism, glomerular filtration rate (GFR), and extracellular (ECFV) and total body fluid volumes (TBFV) of Greyhounds and Beagles. Six healthy Greyhound and six healthy Beagle dogs were studied. Antipyrine, a marker for CYP metabolism and TBFV, and inulin, a marker for the GFR and ECFV, were administered i.v. Samples were collected at predetermined times and plasma was analyzed by validated high-pressure liquid chromatography (HPLC) methods. There were no differences in the disposition or pharmacokinetic parameters for inulin between the dog breeds. However, the clearance of antipyrine (mean = 8.33 mL/min/kg) in Greyhounds was significantly slower than Beagles (13.42 mL/min/kg, P = 0.004). The volume of distribution of antipyrine was significantly larger in Greyhounds (0.789 L/kg) than in Beagles (0.644 L/kg, P = 0.01). The half-life of antipyrine was significantly longer in Greyhounds (1.09 h) compared with Beagles (0.55 h, P = 0.002). The in vitro plasma protein binding of antipyrine was significantly less in Greyhounds (28%) compared with Beagles (40.3%, P = 0.008). Greyhounds exhibited significantly slower CYP metabolism, higher TBFV, and lower in vitro protein binding of antipyrine compared with Beagles. No differences in GFR or ECFV were found.