Simple isocratic high-performance liquid chromatographic method for the separation of the six vitamers of vitamin B6

A simple, sensitive and fast isocratic high-performance liquid chromatographic method has been developed for the separation of all six biologically active forms (vitamers) of vitamin B6. The separation is accomplished using a strong cation-exchange column and a mobile phase of 0.1 M ammonium dihydrogenphosphate adjusted to pH 4.0. All six vitamers are separated within 20 min at a flowrate of 1 ml/min. The concentration of the vitamers is determined with a fluorescence detector (excitation 290 nm; emission 389 nm). The within-run precision of the method expressed as the coefficient of variation is below 5% at the 25 pmol level. Pyridoxal 5'-phosphate can be determined using either pre- or post-column derivatization with sodium bisulfite. Application of the method to cell-free yeast culture media is presented.     

Determination of vitamin B6 using high-performance liquid chromatography with electrochemical detection

A chromatographic method for the determination of vitamin B₆ utilizing a thin-layer amperometric detector with a glassy carbon electrode has been described. The redox behavior of vitamin B₆ has been studied by means of cyclic voltammetry. The limit of detection is 4 ng for pyridoxine and 1 ng for pyridoxamine. Application of the technique for detection of vitamin B₆ in drugs was illustrated.     

A multicommuted fluorescence-based sensing system for simultaneous determination of Vitamins B2 and B6

A multicommuted flow-through optosensor based on the direct fluorescence measurements of Vitamins B₂ and B₆ using a non-polar sorbent (C₁₈ silica gel) as solid sensing zone (to accomplish the separation and subsequent preconcentration/detection of the target analytes) have been developed. The proposed flow system was controlled by Java-written home-made software and designed using three-way solenoid valves for independent automated manipulation of sample and carrier solutions. The native fluorescence signal was simultaneously monitored at two pairs of excitation/emission wavelengths (450/519 and 294/395 for B₂ and B₆, respectively). The separation of the analytes was performed in the detection flow cell, using the differences in the sorption/elution process on the solid support between the two vitamins, due to their different polarity. Using an optimised sampling time, the analytical signal showed linearity in the range 0.01-0.4 and 0.15-3 μg ml⁻¹ with detection limits of 0.003 and 0.045 μg ml⁻¹ for B₂ and B₆, respectively, obtaining R.S.D. (%) values better than 2% for both analytes. The proposed methodology was applied to different pharmaceutical preparations, obtaining remarkably good results with recoveries ranging from 96 to 107.5%.     

Simple high-performance liquid chromatographic method for the determination of medroxyprogesterone acetate in human plasma

The high-performance liquid chromatographic (HPLC) method was developed as a simple, reliable alternative to available methods for measuring plasma concentrations of medroxyprogesterone acetate (MPA). The HPLC method has been successfully automated and is suitable for the rapid, inexpensive analysis of large batches of plasma samples. The best approach involves a solvent extraction followed by HPLC separation and analysis. MPA can be efficiently extracted, at all pH values, by nonpolar solvents. The Spherisorb 5-ODS2 HPLC column provides excellent separation of MPA from endogenous steroids of similar structure and from extraneous plasma blank peaks. A batch of 30-40 samples can be prepared by HPLC analysis in 2-3 hours, with a chromatographic run time of 10 minutes/sample. Calibration curves between 5-250 ng/ml show a good correlation between peak height ratio and MPA concentration, even at low levels. Plasma concentrations of MPA in patients receiving 1 g/day were between 12.6-270 ng/ml in this study, suggesting that the sensitivity of this method, 10 ng/ml, is sufficient for monitoring therapeutic concentrations of MPA. The results show a wide individual variation in plasma concentrations following similar dosing schedules--a finding reported by other workers.     

Simple high-performance liquid chromatographic method for the determination of captopril in biological fluids.

A rapid, simple and sensitive column-switching high-performance liquid chromatographic procedure for the determination of captopril in plasma and urine had been developed. p-Bromophenacyl bromide was used as a derivatizing reagent to react with captopril to form a product that showed ultraviolet-absorbing properties. For plasma samples the protein was removed with 6% perchloric acid before injection. The urine samples were directly injected into the chromatograph. The column-switching system was equipped with a pre-column (5 cm x 0.5 cm I.D.) packed with muBondapak C18 (37-50 microns) and an analytical column (15 cm x 0.5 cm I.D.) packed with YWG-C18, 10 microns. Impurities were washed from the pre-column with 0.2% acetic acid and the retained substances were eluted into the analytical column with acetonitrile-water-acetic acid (35:65:0.4, v/v). Captopril was detected at 260 nm. The calibration curve was linear in the range 20-1000 ng/ml for plasma and 10-200 micrograms/ml for urine. The recoveries averaged 103.2 and 99.5% for plasma and urine, respectively. The coefficients of variation were all less than 10%.     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.     

Simple and rapid high-performance liquid chromatographic method for endogenous alpha-tocopherol determination in human plasma

A simple and rapid reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of endogenous alpha-tocopherol in human plasma. Following addition of alpha-tocopheryl acetate as the internal standard, the plasma was deproteinized using acetonitrile and isopropanol mixture prior to HPLC analysis. Methanol was used as the mobile phase and the effluent was quantitated at 292 nm. By this developed method, the concentrations of alpha-tocopherol were linearly related to their responses in the range of 0.8-30 microg/mL. The relative standard deviations intra-day and inter-day for alpha-tocopherol in plasma were less than 10%. The percentage of bias was within +/-4%, which confirmed the accuracy of the method. The method has been successfully applied for determining endogenous alpha-tocopherol in healthy Thai male volunteers.     

A high-performance liquid chromatographic method for the determination of monofluoroacetate

A simple isocratic high-performance liquid chromatographic (HPLC) method for the quantitative analysis of monofluoroacetic acid (MFA), the toxic substance of Dichapetalum cymosum, in plant material, rumen contents (gastric contents), and liver samples is described. A suitable HPLC column that gives optimum sensitivity, accuracy, precision, and separation of MFA is identified. A C-610 organic acid analysis column at ambient temperature with 0.02M H3PO4 as an eluent and ultraviolet detection at 210 nm is utilized to quantitate MFA. Using this method, the average percentage recovery in plant material, bovine liver, and rumen samples is 94.8%, and a detection limit of 12 microg/L is achievable.     

Comprehensive high-performance liquid chromatographic method for the measurements of lipophilic antioxidants in human plasma

Owing to the increasing interest in the health effects of antioxidant micronutrients on chronic diseases, a robust and rapid HPLC method for simultaneous measurement of coenzyme Q₁₀ (ubiquinone and ubiquinol), vitamin A (all-trans-retinol), vitamin E (tocopherols and tocotrienols) and carotenoids (lutein, zeaxanthin, β-cryptoxanthin, lycopene and β-carotene) was developed. Sample preparation and analytical conditions that would affect solubility and stability of these antioxidants were investigated and optimized. The mobile phase used was made up of acetonitrile, methanol, ethanol and tert-butanol without corrosive additives such as ammonium perchlorate and perchloric acid. Our results show that using two C₁₈ columns coupled with photodiode array, fluorescence and electrochemical detection, a comprehensive spectrum of 16 lipid-soluble antioxidants in 30 μL of plasma could be separated and quantified within 30 min. The chromatographic run time was about 3-fold faster and the sample size was about 5-fold smaller than when assays were performed separately using existing methods. The present method will be useful for dietary habit studies and for antioxidant status investigations.     

Simple reversed-phase liquid chromatography method for determination of tocopherols in edible plant oils

A simple and rapid reversed-phase high-performance liquid chromatography method for determination of alpha-, (beta + gamma), and delta-tocopherols in edible plant oils has been developed. Oils are diluted in 2-propanol and injected directly onto Symmetry C18 column. Methanol and acetonitrile (1:1) are used as a mobile phase. Tocopherols are detected using fluorescence detector set at excitation and emission wavelength 295 nm and 325 nm, respectively. The method is precise (R.S.D. not higher than 2.24%) and sensitive-detection limits (DL) are 8 ng/ml for gamma- and delta-tocopherols and 28 ng/ml for alpha-tocopherol; quantification limits (QL) were calculated as three times higher than DL.     

Rapid high-performance liquid chromatographic method for the determination of peroxyacetic acid

The selective oxidation of methyl p-tolyl sulfide (MTS) to the corresponding sulfoxide (MTSO) by peroxyacetic acid and the subsequent rapid separation of the sulfide and sulfoxide are the basis for a fast and reliable HPLC method for the determination of this oxidizing agent in the presence of hydrogen peroxide. The time required for chromatographic separation was reduced to less than 1 min. To improve the long-term stability of the sulfoxide solution, hydrogen peroxide was decomposed catalytically by manganese dioxide. Even in the presence of a tenfold molar excess of hydrogen peroxide, a storability of at least 20 h without a significant increase in MTSO concentration was achieved. External calibration can be performed using the stable and commercially available MTSO. Real samples from a brewery cleaning-in-place disinfection process were analysed and the results were compared with those of the classical two-step titration.     

Determination of vitamin B12 in multivitamin tablets by multimode high-performance liquid chromatography

Quantitative determination of vitamin B₁₂ in B-complex tablets was performed by using multimode high-performance liquid chromatography. The multivitamin tablets (B₁, B₆ and B₁₂) were sonicated for 30 min in methanol–water (50:50, v/v) and diluted to appropriated volume with the same solvent. The resulting solution was filtered and the filtrate was analysed on a phenylpropanolamine bonded silica column (15 cm×4.6 mm I.D., 5 μm). The optimized mobile phase was 30 mM phosphate buffer (pH 3.00) containing 6% (v/v) acetonitrile at a flow-rate of 1 ml min⁻¹ and the detection was measured at 361 nm. The calibration graph prepared using standards was linear from 0.05 to 0.25 μg. The determination limit was 25 ng, the relative standard deviation was 0.47% and recovery from tablet solution was 100%. An analysis was completed in 5 min. The new method is simple, rapid and precise.     

Optimization of high-performance liquid chromatographic parameters for the determination of capsaicinoid compounds using the simplex method.

A high-performance liquid chromatographic method was developed for the analysis of capsaicinoid compounds, the pungent principles of capsicum fruits. A sequential simplex method was applied to optimize the chromatographic response function used to assess the quality of separation by varying the chromatographic parameters. The separation was achieved in 11 min using a C-8 column of 15-cm length and 4.6 mm diameter using a UV detector. A flow rate of 1.15 ml min(-1) at a column temperature of 43.5 degrees C using 63.7% methanol in water gave the most efficient separation. The method was found to be suitable for the determination of the major capsaicinoid compounds in the capsicum samples.     

Simple high-performance liquid chromatographic method for determination of atropine and obidoxime in a parenteral injection device

Atropine and obidoxime in a parenteral injection device are determined by simple HPLC method simultaneously without any pretreatment at 228 nm. The relative standard deviations (R.S.D.) were below 1.6% for the compounds. The correlation coefficient was greater than 0.999 for both compounds in the calibration range. The recoveries at 5 mg/L concentration averaged as 95% for atropine and 102% for obidoxime. The uncertainty of the measurements for atropine and obidoxime was 2.8% and 2.4%, respectively.     

Simple high-performance liquid chromatographic method for the measurement of amiloride in body fluids

A simplified rapid high-performance liquid chromatographic (HPLC) procedure has been developed for the measurement of amiloride in plasma or urine. Solid-phase extraction columns provide quick, clean and simple sample preparation, allowing ten samples to be processed in less than 5 min. The HPLC system uses a standard reversed-phase (C18) column with detection by ultraviolet absorption at 365 nm. The assay has been used to define plasma amiloride concentration-time profiles and to quantitate urine amiloride recovery in healthy men after repeated administration at two doses.