Optimum Column Temperature Research Articles

Development of a New, Fully Validated LC-MS/MS Method for the Analysis of Flibanserin in Pharmaceutical Preparations and Comparison of the Chromatographic Performance with Six Stationary Phase Types

Background: Initially synthesized as an antidepressant and potentially rapid onset of action, flibanserin (FLB) was approved by the Food and Drug Administration (FDA) in August 2015 with a warning to dispense the drug through a dedicated risk management program, despite the removal of HSDD from the DSM-5TM. The drug is the first noteworthy FDA-approved drug for treating premenopausal women with acquired, generalized HSDD. Objective: In literature, studies are plasma analyses or metabolite determinations to meet pharmacokinetic analyses and some analytical targets. For this reason, in this thesis, a new method has been developed for analysing FLB from pharmaceutical preparations, which is our target, providing all optimization conditions and method validity parameters. Methods: The chromatographic separation was also investigated using Chromolith® and Ascentis® Express models with a total of six stationary phases. The mobile phase mixture was acetonitrile:ammonium formate (0.020 M, pH 6.0) and was used at the ratio (60:40, v/v). The optimum column temperature was chosen as 40.0±0.1°C, and the autosampler thermostat temperature was chosen as 15±0.1°C. The sample injection volume is optimized to be 1 μL. Results: The developed method is linear in the range of 2.63–105.0 ng/mL, and the regression coefficient is 0.999 intraday and 0.986 interday. In the method, LOD and LOQ were obtained as 128 pg/mL and 384 pg/mL, respectively. In addition, the ANOVA P values were calculated as 0.586 and 0.914, respectively, in the validation studies conducted intraday and interday. Conclusion: FLB chromatographic behaviors were studied and compared in detail with six different stationary phases. The developed method was fully validated according to the ICH Q2 (R1) guideline, and its pseudo-pharmaceutical formulation was analyzed.

Development and Validation of an RP-HPLC Method for Determination of Solasodine, A Steroidal Alkaloid

: Objective: The aim of the present study was to optimize an RP-HPLC method for determination of solasodine, a steroidal alkaloid with various pharmacologic properties, in herbal medicinal preparations. Methods: Various chromatographic parameters including the composition of mobile phase and its organic and pH modifiers, mode of elution and column temperature were investigated using analytical C18 columns as stationary phase. Acetonitrile and methanol were examined in combination with deionized water and different aqueous buffers as mobile phases. Results: A mixture of methanol:KH2PO4 buffer (pH 2.5) at the ratio of 75:25 %v/v in an isocratic elution mode produced more symmetric, sharper peaks with better retention times and capacity factor than that of acetonitrile:KH2PO4 buffer. The optimum column temperature was found at 25°C. The standard calibration curve showed a very good linearity (R2=0.9966) in the range of 1-25 μg/ml with acceptable inter-day and intra-day precisions and accuracy. In addition, the LOD and the LOQ of the method were determined at 0.2 and 0.7 μg/mL, respectively. Conclusion: This developed and validated method is simple and quite rapid and can be used for routine determination of solasodine in samples such as traditional medicinal preparations.Key words: Solasodine, Steroidal alkaloid, RP-HPLC, Validation.

Improvement in HPLC separation of acetic acid and levulinic acid in the profiling of biomass hydrolysate

5-Hydroxymethylfurfural (HMF) and furfural could be separated by the Aminex HPX-87H column chromatography, however, the separation and quantification of acetic acid and levulinic acid in biomass hydrolysate have been difficult with this method. In present study, the HPLC separation of acetic acid and levulinic acid on Aminex HPX-87H column has been investigated by varying column temperature, flow rate, and sulfuric acid content in the mobile phase.The column temperature was found critical in resolving acetic acid and levulinic acid. The resolution for two acids increased dramatically from 0.42 to 1.86 when the column temperature was lowered from 60 to 30°C. So did the capacity factors for levulinic acid that was increased from 1.20 to 1.44 as the column temperature dropped. The optimum column temperature for the separation was found at 45°C. Variation in flow rate and sulfuric acid concentration improved not as much as the column temperature did.

THE EFFECT OF DISTILLATE STORING DISTILLED FROM FRUCTOSE SYRUPS TOWARD ITS ACETALDEHYDE CONCENTRATION MEASURED BY GAS CHROMATOGRAPHY

Acetaldehyde is a compound of aldehyde group that is very volatile and toxic. This compound can be found in fructose syrups used in carbonate beverages. The syrups had been distilled then analysed using gas chromatography. The concentration of acetaldehyde was 289.78 g/g in the distillates kept for one week, 295.30 g/g in those kept for two weeks, 429.45 g/g in those kept for three weeks, and 449.38 g/g in those kept for four weeks. The optimum column temperature was programmed with initial temperature of 40 oC held on for four minutes, then increasing by 40 oC/minute to 200 oC. It can be concluded that the longer the distillates have been kept, the greater the concentration of acetaldehyde in the distillates. A further research to investigate the present of microbe in the distillates and the effect of pH should be conducted Keywords: acetaldehyde, fructose syrup, distillates, gas chromatography.

DNA sequencing up to 1300 bases in two hours by capillary electrophoresis with mixed replaceable linear polyacrylamide solutions.

This paper presents results on ultralong read DNA sequencing with relatively short separation times using capillary electrophoresis with replaceable polymer matrixes. In previous work, the effectiveness of mixed replaceable solutions of linear polyacrylamide (LPA) was demonstrated, and 1000 bases were routinely obtained in less than 1 h. Substantially longer read lengths have now been achieved by a combination of improved formulation of LPA mixtures, optimization of temperature and electric field, adjustment of the sequencing reaction, and refinement of the base-caller. The average molar masses of LPA used as DNA separation matrixes were measured by gel permeation chromatography and multiangle laser light scattering. Newly formulated matrixes comprising 0.5% (w/w) 270 kDa and 2% (w/w) 10 or 17 MDa LPA raised the optimum column temperature from 60 to 70 degrees C, increasing the selectivity for large DNA fragments, while maintaining high selectivity for small fragments as well. This improved resolution was further enhanced by reducing the electric field strength from 200 to 125 V/cm. In addition, because sequencing accuracy beyond 1000 bases was diminished by the low signal from G-terminated fragments when the standard reaction protocol for a commercial dye primer kit was used, the amount of these fragments was doubled. Augmenting the base-calling expert system with rules specific for low peak resolution also had a significant effect, contributing slightly less than half of the total increase in read length. With full optimization, this read length reached up to 1300 bases (average 1250) with 98.5% accuracy in 2 h for a single-stranded M13 template.

Precalculation of the optimum column temperature for gas chromatographic separation of petroleum fractions

Determination of the optimum column temperature is very time-consuming when a new gas chromatographic method is set up. An algorithm is proposed for precalculation of the optimum temperature. It is based on two concepts, the so-called unified retention index and a mathematical description of dependence of peak widths on the retention indices and on column temperature. By means of a defined object function and a scanning optimization strategy, a program is written in BASIC. Several different problems are considered, including the optimum temperature for calculation of the composition of n- and iso-alkanes, cycloalkanes, alkenes and arenes in a petroleum fraction, determination of individual contents and of key components, and separation at any given temperature. The possibilities of the program are illustrated by three examples.

Liquid-chromatographic determination of tobramycin in serum with spectrophotometric detection.

We describe a simple, precise, accurate, and specific liquid-chromatographic procedure for determination of tobramycin in 50 microL of serum. Tobramycin and the internal standard (sisomicin) are quantitatively converted into their trinitrophenyl derivatives by reaction with a water-soluble derivatizing agent (2,4,6-trinitrobenzenesulfonic acid) at 70 degrees C for 30 min. The derivatives are extracted from the crude reaction mixture by using a reversed-phase Bond-Elut C18 column, and separated on a reversed-phase octyl column with a mobile phase consisting of an acetonitrile/phosphate buffer (70/30 by vol) at a flow rate of 3.0 mL/min. The eluted compounds are detected at 340 nm, and quantified from their peak areas. Chromatography is complete in less than 4.5 min at the optimum column temperature of 50 degrees C. The lower limit of detection for tobramycin is less than 0.2 mg/L. Analytical recoveries for tobramycin varied from 94 to 99%, linearity extended to 25 mg/L, and day-to-day precision (CV) was between 4.6 and 5.1%. Numerous drugs and antibiotics tested do not interfere. Results correlate well (r greater than 0.95) with those by radioimmunoassay and EMIT.

Rapid method for screening toxic drugs in serum with liquid chromatography.

We present a method for the simultaneous analysis of a variety of commonly abused drugs (acetaminophen, theophylline, salicylate, primidone, methyprylon, phenobarbital, butabarbital, ethchlorvynol, butalbital, chlordiazepoxide, pentobarbital, amobarbital, phenytoin, glutethimide, secobarbital, flurazepam, nitrazepam, methaqualone, N-desmethyldiazepam, and diazepam) in serum or plasma. Serum proteins are precipitated with an acetonitrile solution containing hexobarbital, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of acetonitrile/phosphate buffer (pH 3.2), using a two-step linear gradient, at a flow rate of 3.0 mL/min. The eluted drugs are detected by their absorption at 210 nm; their quantities are estimated from their peak heights. A complete analysis requires no longer than 45 minutes at the optimum column temperature of 50 degree C. A sensitivity of 2 mg/L of serum is attained routinely for most of the hypnotic and analgesic drugs; while methaqualone, chlordiazepoxide, diazepam, and N-desmethyldiazepam can be detected at a concentration of 0.2 mg/L. Analytical recoveries for the twenty drugs varied from 93-112%, with good reproducibility. Of more than forty drugs tested for possible interference, desmethyldoxepin, procainamide, phenylpropanolamine, mesantoin, and phenacetin interfere with the analysis of flurazepam, acetaminophen, ethchlorvynol, and phenobarbital, respectively.

Improved liquid-chromatographic method for determination of serum cortisol.

We describe a specific and precise method for measuring concentrations of cortisol in serum or plasma by liquid chromatography. Cortisol, together with an internal standard, equilenin, is extracted from 1 mL of serum or plasma and analyzed isocratically on a reversed-phase column with a mobile phase of acetonitrile/phosphate buffer (30/70, by vol.), at a flow rate of 2.0 mL/min. The eluted cortisol is detected by its absorption at 254 nm and quantitated by peak height measurements. Each analysis requires no longer than 15 min at the optimum column temperature of 50 degrees C. The lower limit of detection for cortisol is about 2 ng/sample for a standard solution; sensitivity is routinely 5 micrograms/L of serum. Analytical recoveries exceeded 95%, with good day-to-day precision (coefficients of variation between 4 and 7%). Of more than 50 drugs and steroids tested for possible interference, only the steroids cortisone, prednisone, and prednisolone may interfere with the analysis of cortisol.

A Simultaneous High-Performance Liquid Chromatographic Analysis of the Most Common Anticonvulsants and Their Metabolites in the Serum

we present a method for the simultaneous analysis of some common anticonvulsants and their metabolites (phenylethyl malonamide, primidone, ethosuximide, 5-ethyl-5-phenylhydantoin, phenobarbital, tx-methyl-ct-phenylsuccinlmide, mephenytoin, methsuximide, mephobarbital, phenytoin, and carbamazepine) in as little as 25/JI of serum. Serum proteins are precipitated with an acetonitrile solution containing 5-(4methylphanyl)-5-phenylhydantoin, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer, at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm; their quantities are estimated from their peak heights. Each analysis requires no longer than 30 minutes at the optimum column temperature of 50C. The lower limit of detection for all these drugs is less than 10 ng/sample for drug standards. A sensitivity of 1.0 mglllter of serum is attained routinely for each of the drugs. Analytical recoveries for the eleven drugs varied from 88 to 102%, with good day-to-day precision (CV, between 2.0 and 8.0%). Of more than 35 drugs tested for possible Interference, only ethotoin and pentobarbital interfere with the analysis of phenobarbital and mephobarbital, respectively.

Simultaneous liquid-chromatographic determination of 12 common sedatives and hypnotics in serum.

We present a method for simultaneously determining 12 hypnotics and sedatives (primidone, methyprylon, phenobarbital, butabarbital, butalbital, ethchlorvynol, pentobarbital, amobarbital, phenytoin, glutethimide, secobarbital and methaqualone) in 200 microliter of serum. Serum proteins are precipitated with an acetonitrile solution containing 5-(4-methylphenyl)-5-phenylhydantoin, the internal standard. The drugs are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer, at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm; their quantities are estimated from their peak heights. Each analysis requires no longer than 30 min at the optimum column temperature of 50 degrees C. The lower limit of detection for all of these drugs is less than 10 ng/sample for drug standard. A sensitivity of 1.0 mg/liter of serum is attained routinely for each of the drugs. Analytical recoveries for the 12 drugs varied from 94 to 112%, with good day-to-day precision (CV between 3.8 and 10.4%). Of more than 35 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.

Optimization of operating parameters for glass capillary column gas chromatography

The relationship between analysis time and separation with glass capillary columns was studied. Optimal operating parameters for achieving the shortest analysis times consistent with degree of separation were determined experimentally. It was found that the optimum column temperature is governed by retention characteristics of the solutes and the optimum column length by the required magnitude of separation. The optimum carrier gas flow-rate was found to be a function of the column length. Emphasis is placed on using capillary column length as an operating parameter.

Simultaneous measurement of phenobarbital, phenytoin, primidone, ethosuximide, and carbamazepine in serum by high-pressure liquid chromatography.

We present a method for simultaneously determining five anticonvulsants [phenobarbital, phenytoin (diphenylhydantoin), primidone, ethosuximide, and carbamazepine] in as little as 25 microliters of serum. The proteins are precipitated with an acetonitrile solution containing hexobarbital as an internal standard. The anticonvulsants are eluted from a reversed-phase column with a mobile phase consisting of an acetonitrile/phosphate buffer (19/81 by vol) at a flow rate of 3.0 ml/min. The eluted drugs are detected by their absorption at 195 nm, and quantities estimated from their peak heights. Each analysis requires about 14 min. at an optimum column temperature of 50 degrees C. The lower unit of detection for all of these drugs is less than 10 ng. Sensitivities, for serum samples, of 1.0 mg/liter for all the drugs analyzed are attained routinely. Analytical recoveries for the five drugs varied from 97 - 107%, with good day-to-day precision (CV between 3.9 and 5.9%). Of more than 30 drugs tested for possible interference, only ethotoin interferes with the analysis of phenobarbital.

Rapid micromethod for measuring anticonvulsant drugs in serum by high-performance liquid chromatography.

We describe an assay system for measuring phenobarbital, diphenylhydantoin, primidone, ethosuximide, and carbamazepine in 25 mul of serum. The procedure involves precipitation of proteins with an acetonitrile solution containing cyheptamide as an internal standard, and reverse-phase chromatography on a 4 mm X 30 cm column containing "muBondapak C18." The anticonvulsants are eluted with an equivolume mixture of potassium phosphate buffer (10 mmol/liter, pH 8.0) and acetonitrile at a flow rate of 0.8 ml/min, detected by their absorbance at 200 nm, and quantitated by measuring peak areas. When measurement of primidone is not required, a 254-nm detector may be used. Each analysis requires 10 min. Optimum column temperature has been found to decrease with use. Analytical recoveries for the five drugs varied from 92% to 102% with good precision (coefficients of variation between 2.8% and 9.2% for therapeutic and toxic concentrations). The results obtained with this method compare favorably with results obtained by gas-liquid chromatography (correlation coefficients between 0.77 and 0.98). In over 1300 patients' samples analyzed to date, the only drugs known to have interfered with the assay are gentamicin, diazoxide, and mephobarbital.

Feed concentration and column temperature in production and preparative chromatography

For large scale chromatographic separations, economic operation will require high concentrations of the feed material in the mobile phase entering the column. The different types of ultimate concentration limit encountered in liquid and gas chromatography are described. When separating thermally stable materials with a gas—liquid column, an optimum column temperature exists where the throughput at the ultimate concentration limit goes through a maximum. Equations are derived for calculating the optimum temperature and throughput. For heat-sensitive materials the column should be operated at the optimum temperature or the degradation temperature, whichever is the lower. The effect of temperature on the length of column required for separation is described. The treatment is made possible by taking the width and concentration of the feed band as roughly independent parameters, a satisfactory approximation when wide bands are used.

Gas-liquid partition chromatography : Column behavior and its reproduction criteria for favorable column behavior

The distribution of a solute in a gas-liquid Chromatographic column has been discussed from the probability view point and its analogy to the Bernoulli trial system has been shown. It has been shown that the reproduction of column behavior is possible by reproducing the probability. The criterion for the optimum column temperature has also been discussed. Recommendations have been made for determining the optimum column conditions and its reproduction.