Two high-performance liquid chromatographic methods for quantitation of theophylline in plasma

Two high-performance liquid chromatographic methods are described for the assay of theophylline in plasma. Both allowed the separation of theophylline from the caffeine metabolites, theobromine and 1,7-dimethylxanthine. Method A, using 8-chlorotheophylline as internal standard, involved back extraction of theophylline from organic extract with 0.1 M sodium hydroxide. Method B used generally accepted solvent extraction followed by evaporation and beta-hydroxyethyltheophylline as internal standard. High-performance liquid chromatographic analyses were performed on reversed-phase phenyl columns (25 X 0.46 and 25 X 0.41 cm) using 20% methanol in 20 mM phosphate buffer at pH 5.6 for Method A and 2% acetonitrile and 8% methanol in 20 mM phosphate buffer for Method B. The column effluent was monitored at UV 273 nm. Standard curves for both Methods A and B were fitted by linear regression (r greater than 0.999) in the concentration range of 0.05-50 micrograms/ml. Either method was selective, accurate and reproducible over the concentration range 0.08-26 micrograms/ml. However, compared with Method B, Method A provided significant advantages in terms of simplicity, speed and efficiency.     

Validation of a strategic approach to high-performance liquid chromatographic method selection

Summary A recently reported chromatographic method selection strategy has been validated using fifteen drug formulations selected randomly from the Belgium Compendium, 1992. On the basis of the hydrophobic and the acidic — basic properties of the sample components, reversed-phase was recommended as the first choice mode for all formulations. For two multicomponent formulations the range of analyte polarity dictated the need for gradient elution. The commerical software DryLab G/plus^® was used for selection of optimum gradient conditions. The results obtained by both isocratic and gradient chromatography are discussed, as is the usefulness of a tailing suppressor in both modes.     

A high-performance liquid chromatographic method for saikosaponin a quantification in rat plasma

A high-performance liquid chromatographic method with UV detection has been developed for the determination of saikosaponin a in rat plasma. Saikosaponin a and internal standard jujuboside A were isolated from plasma samples by solid-phase extraction. The chromatographic separation was achieved on a reversed-phase C(18) column with the mobile phase of acetonitrile-water (35:65, v/v) at a flow rate of 1 mL/min and UV detection was set at 205 nm. The standard curve for saikosaponin a was linear over the concentration range 0.25-10 microg/mL and the limit of detection was 0.05 microg/mL. The absolute recovery was greater than 82%. The precision and accuracy ranged from 3.05 to 9.59% and 95.61 to 110.00%, respectively. The validated method was used to determine saikosaponin a in plasma samples in a pharmacokinetic study of saikosaponin a administered to Sprague-Dawley rats.     

Enantioselective high-performance liquid chromatographic method for the determination of baclofen in human plasma

A new analytical method for the separation and determination of R-(−)- and S-(+)- baclofen enantiomers in human plasma by high-performance liquid chromatography (HPLC) with UV detection was developed. Enantioselective resolution of the baclofen enantiomers was achieved by using teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol: glacial acetic acid: triethylamine, 100:0.1:0.1, (v/v/v) at a flow rate of 0.5 ml min⁻¹ and UV detection set at 220 nm. The analytes of interest with S-(+)-sulpiride as the internal standard were extracted from human plasma using liquid-liquid extraction procedure with ethyl ether under alkaline condition prior to HPLC analysis. Recoveries for R-(−)- and S-(+)-baclofen enantiomers were in the ranges of 96-103% at 60-2500 ng ml⁻¹ level. Intra-day and inter-day precision calculated as %RSD was in the ranges of 1.2-5.2 and 1.3-4.3% for both enantiomers, respectively. Intra-day and inter-day accuracy calculated as percentage error were in the ranges of 1.2-3.9 and 1.1-3.9% for both enantiomers, respectively. Linear calibration curves in the concentration ranges of 20-3000 ng ml⁻¹ for each enantiomer showed correlation coefficient (r) of 0.9997. The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 20 and 10 ng ml⁻¹ (S/N=3) respectively.     

A high performance liquid chromatographic method for the quantitation of flecainide in plasma.

A routine high performance liquid chromatographic method for the rapid determination of fleca?nide (Flecaine), using a novel internal standard, N-methylfleca?nide, has been developed. After deproteinization of spiked samples, fleca?nide was totally recovered at neutral pH. Fleca?nide and the internal standard were separated on a reversed phase XL 3 microns ODS column using 10 mM phosphate buffer, pH 3.0: acetonitrile (70:30) as mobile phase, in less than 10 min. With spectrofluorometric detection, the limit of quantitation for fleca?nide was 10 ng/mL. Intra- and inter-assay precision variations were 0.24% and 1.4%.     

Validation of a high-performance liquid chromatographic-radioimmunoassay method for the determination of lacidipine in plasma.

A sensitive and reproducible method for the determination of lacidipine, a new potent antihypertensive dihydropyridine, is reported. The method involves solid-phase extraction, reversed-phase high-performance liquid chromatography and radioimmunoassay of the collected fraction. The assay provides a limit of detection of 20 pg/ml of plasma, allowing the determination of trough (24 h) plasma concentrations. The method is suitable for pharmacokinetic studies in man.     

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.     

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.     

Validation of a column liquid chromatographic method for phytate

A column liquid chromatographic method that uses a spectrophotometric detector for the analysis of phytate and other inositol phosphates in foodstuffs and seeds is described. It has been tested thoroughly and sent to 6 other laboratories where there was an interest in such an analytical method and the equipment was available for performing it. These samples were blinded to the individual collaborators. Also sent was a standard wheat bran sample available from the American Association of Cereal Chemists with a standard phytate value reported as 3.2%. The postcolumn sulfosalicylic acid reaction was developed in the 1950s as an analytical method for ferric ion. All reagents are aqueous solutions and the specific pH values of each are important. The column separation is at pH 4, which dissociates all phosphate analogs. The ferric sulfosalicylate is at pH 1.8. The postcolumn reaction is then at pH 2.0-2.3. At this pH, the sulfosalicylate complexes one ferric ion and will yield it to a stronger complexing agent such as a phosphate ester. At higher pH values, sulfosalicylate complexes 2 ferric ions and will yield neither to these anions. The method sensitivity is 8-12 nmoles/injection.     

Validation of a solid-phase extraction high-performance liquid chromatographic assay for doxazosin.

The determination of doxazosin by high-performance liquid chromatography with fluorescence detection is described. Propanolol was used as the internal standard. Plasma samples were treated with methanol to precipitate the proteins. Doxazosin was isolated with C18 reversed-phase extraction columns. The determination limit is 1 ng/ml of plasma, while the extraction columns can be reused frequently. The method is applied to clinical trial samples.     

A validated new method for nevirapine quantitation in human plasma via high-performance liquid chromatography

A fully validated and clinically relevant assay was developed for the assessment of nevirapine concentrations in neonate blood plasma samples. Solid-phase extraction with an acid-base wash series was used to prepare subject samples for analysis. Samples were separated by high performance liquid chromatography and detected at 280 nm on a C8 reverse-phase column in an isocratic mobile phase. The retention times of nevirapine and its internal standard were 5.0 and 6.9 min, respectively. The method was validated by assessment of accuracy and precision (statistical values <15%), specificity, and stability. The assay was linear in the range 25-10,000 ng/mL (r2 > 0.996) and the average recovery was 93% (n = 18). The lower limit of quantification (relative standard deviation <20%) was determined to be 25 ng/mL for 50 microL of plasma, allowing detection of as little as 1.25 ng of nevirapine in a sample. This value represents an increase in sensitivity of up to 30-fold over previously published methods.