Improved high-performance liquid chromatographic assay method for the enantiomers of ibuprofen

A rapid, inexpensive and sensitive high-performance liquid chromatographic method for the quantitation of ibuprofen enantiomers from a variety of biological fluids is reported. This method uses a commercially available internal standard and has significantly less interference from endogenous co-extracted solutes than do previously reported methods. The method involves the acid extraction of drug and internal standard [(±)-fenoprofen] from the biological fluid with isooctane—isopropanol (95:5) followed by evaporation and derivatization with enthylchloroformate and R-(+)-α-phenylethylamine. Excellent linearity was observed between the peak-area ratio and enantiomer concentration (r > 0.99) over a concentration range of 0.25–50 μg/ml. This method is suitable for the quantitation of ibuprofen from single-dose pharmacokinetic studies involving either rats or humans.     

High-performance thin-layer chromatographic determination of ibuprofen in plasma

A high-performance thin-layer chromatographic (HPTLC) method for quantitation of ibuprofen from plasma is described. The drug was extracted from acidified plasma with hexane-isopropanol (85:15). The mobile phase composition was n-hexane-ethyl acetate-anhydrous acetic acid (75:25:2). Densitometric analysis of ibuprofen was carried out at 222 nm. The calibration curves of ibuprofen in chloroform and in plasma were linear over the range 2–20 μg. The mean values of intercept, slope and correlation coefficient were 0.0422±0.0018, 0.0356±0.0213 and 0.9976±0.0013 for standard curves in chloroform and 0.1044±0.003, 0.8759±0.0213 and 0.9939±0.001 for standard curves in plasma, respectively. The limit of detection of ibuprofen from human plasma (assay sensitivity) was 50 ng and no interference was found from endogenous compounds. The recovery of ibuprofen from human plasma using the described extraction procedure was about 85%. The mean relative standard deviations for within-day and between-day analyses were 2.24 and 2.6% for 5 μg and 3.67 and 3.2% for 15μg ibuprofen concentration, respectively. The method was utilized to monitor the plasma concentration of ibuprofen post administration of sustained release capsules in human patient volunteers.     

Validation of a liquid chromatographic method for the determination of ibuprofen in human plasma

A method based on LC-MS-MS is described for the determination of methyldopa in human plasma using dopa-phenyl-D3 as the internal standard. The method has a chromatographic run time of 5.5 min and was linear in the range of 20-5000 ng/ml. The limit of quantitation was 20 ng/ml, the intra-day precisions were 7.3, 5.4 and 4.3% and the intra-day accuracies were -8.0, -1.3 and -2.0% for 30, 600 and 3000 ng/ml, respectively. The inter-day precisions were 7.7, 0.5 and 0.7% and the inter-day accuracies were 0.2, -1.1 and -2.3%, respectively, for the above concentrations. This method was employed in a bioequivalence study of two tablet formulations of methyldopa.     

Validation of a high-performance liquid chromatographic method for the determination of doxycycline in turkey plasma

A high-performance liquid chromatographic method for the analysis of doxycycline in turkey plasma samples using demeclocycline hydrochloride as the internal standard was developed, optimized and validated, A one-step extraction procedure and an isocratic HPLC method with UV detection were used. No interferences with endogenous compounds or with the anticoagulant were observed, Linear calibration curves (r²>0.99) were obtained in water and plasma between 0 and 600 μg ml⁻¹. Good recoveries for doxycycline (>66%) and demeclocycline (>72%) were seen both in water and in plasma, The coefficient of variation was <9.86% for within-day reproducibility and <7.53% for the between-day reproducibility. The deviation between the mean value found and the true value was <14.5% (accuracy). The limit of detection was 0.1 μg ml⁻¹ in plasma samples. A good stability of doxycycline was observed in water and in plasma samples after storage for six months at −20°C (recovery >91%).     

A new high-performance liquid chromatographic method for determination of warfarin enantiomers

Warfarin is the most common agent used for control and prevention of venous as well as arterial thromboembolism. Although warfarin is administered as a racemic mixture of two stereoisomers (S and R), the S-form is mainly responsible for the anticoagulant effect. The anticoagulant effect of the drug is monitored by analysis of prothrombin complex (International Normalised Ratio,INR). In some cases, however, the measurements of plasma warfarin concentration are needed. Here, we present a new, rapid, sensitive and cost-effective HPLC-method for the determination of warfarin enantiomers in plasma. The chromatographic system consisted of Waters 616 gradient pump, Waters 996 photo diode array detector, Gilson 230 autoinjector and Pirkle (R,R) Whelk-O1 column (25 cmx4.6 mm I.D., 5 microm). An isocratic mobile phase of methanol/acetonitrile/water (50/10/40, v/v) with 0.1% glacial acetic acid was used. The follow rate was 1 mL/min. Data analysis was carried out with Waters Millennium32. The absorbance at 305 nm was measured with a total run-time of 15 min. Method linearity was studied by establishing regression data containing eight points over the range 0.08-10 microg/mL. In this range, warfarin showed to be linear (r2=0.9997 for S-warfarin and r2=0.9998 for R-warfarin). The limit of detection in plasma was 16 ng/mL for S-warfarin and 18 ng/mL for R-warfarin. Limit of quatitation was defined as 10xLOD. The extraction recovery was approximately 80%. Also the relation between INR and warfarin concentration was investigated. As expected, there was a low correlation between these two variables (r=0.23, y=0.3044x+0.9712). This method offers a rapid and cost-effective determination of warfarin enantiomers in human plasma.     

Sensitive high-performance liquid chromatographic method for the determination of coumarin in plasma

A high-performance liquid chromatographic method was developed for the determination of coumarin in plasma at low concentrations. The method involves a single-step extraction of the alkalinized sample with hexane and subsequent evaporation of the organic phase in the presence of hydrochloric acid to collect and concentrate the coumarin. Analysis of the acidic phase was performed on a C₈ column and coumarin was detected by measuring the UV absorbance at 275 nm. The limit of detection was 0.3 μg l⁻¹. The assay was used to study the evolution of concentrations of coumarin in one volunteer after oral administration of a single 10-mg dose.     

Automated high-performance liquid chromatographic method for determination of furosemide in dog plasma

An automated high-performance liquid chromatographic method for the determination of the diuretic drug furosemide has been established. Dog plasma was injected directly into a two-column system with a BSA—ODS (ODS column coated with bovine serum albumin) precolumn and a C₁₈ analytical column for the separation of furosemide. The two columns were automatically switched. Furosemide remained trapped on the precolumn while proteins were eluted to waste. After column switching, furosemide was washed onto the analytical column and analysed without interference. The greatest advantage of the method is its easy performance without manual sample preparation; it requires no extraction or deproteinization. The method allows determination of 0.1–10 μg/ml of furosemide with accuracy and precision comparable with previously reported values. The coefficients of variation obtained from replicate measurements of 1 μg/ml and 5 μg/ml samples were 1.65% and 2.40%, respectively. This method was used to measure the plasma levels of furosemide in beagle dogs to whom the drugs was administered, as a reference, in a toxicological study.     

Simple high-performance liquid chromatographic method for determination of ketoconazole in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of ketoconazole in human plasma. The method entailed direct injection of the plasma sample after deproteinization using acetonitrile. The mobile phase comprised 0.05 M disodium hydrogen orthophosphate and acetonitrile (50:50, v/v) adjusted to pH 6. Analysis was run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The method is specific and sensitive with a quantification limit of approximately 60 ng/ml and a detection limit of 40 ng/ml at a signal-to-noise ratio of 3:1. Mean absolute recovery value was about 105%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 14%. The calibration curve was linear over a concentration range of 62.5–8000 ng/ml.     

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma

A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.     

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

A simple high-performance liquid chromatographic method using ultraviolet detection was developed for the determination of metformin in human plasma. The method entailed direct injection of the plasma sample after deproteination using perchloric acid. The mobile phase comprised 0.01 M potassium dihydrogen orthophosphate (pH 3.5) and acetonitrile (60:40, v/v). Analyses were run at a flow-rate of 1.0 ml/min with the detector operating at a detection wavelength of 234 nm. The method is specific and sensitive, with a quantification limit of approximately 60 ng/ml and a detection limit of 15 ng/ml at a signal-to-noise ratio of 3:1. The mean absolute recovery value was about 97%, while the within-day and between-day coefficient of variation and percent error values of the assay method were all less than 8%. The calibration curve was linear over a concentration range of 62.5–4000 ng/ml.     

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

A simple high-performance liquid chromatographic method was developed for the determination of ranitidine in human plasma. Prior to analysis, ranitidine and the internal standard (metoprolol) were extracted from alkalinized plasma samples using dichloromethane. The mobile phase was 0.05 M potassium dihydrogenphosphate–acetonitrile (88:12, v/v) adjusted to pH 6.5. Analysis was run at a flow-rate of 1.3 ml/min and at a detection wavelength of 229 nm. The method is sensitive with a detection limit of 1 ng/ml at a signal-to-noise ratio of 3:1, while the quantification limit was set at 15 ng/ml. The calibration curve was linear over a concentration range of 15–2000 ng/ml. Mean recovery value of the extraction procedure was about 90%, while the within-day and between-day coefficients of variation and percent error values of the assay method were all less than 15%.     

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

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.     

Quantitative determination of naproxen in plasma by a simple high-performance liquid chromatographic method

A high-performance liquid chromatographic method for the determination of naproxen in plasma is described. The technique is based on the single extraction of the drug from acidified plasma with chloroform using 2-naphthalene acetic acid as internal standard. The chromatographic system consisted of a column packed with Spherisorb ODS (5 μm); the mobile phase was acetonitrile—phosphoric acid (pH 3) (45:55, v/v).The method can accurately measure plasma naproxen concentrations down to 1 μg/ml using 100 μl of sample, with no interference from endogenous compounds. The coefficients of variation of the method at 120 μg/ml and 1 μg/ml are 2.8 and 21.6%, respectively, and the calibration curve is linear. The method described is very suitable for routine clinical and pharmacokinetic studies.     

Liquid chromatographic determination of the enantiomers of ibuprofen in plasma using a chiral AGP column

A reversed-phase high-performance liquid chromatographic method has been developed for the determination of the R- and S-enantiomers of ibuprofen. The enantiomers and the internal standard 4-pentylphenylacetic acid are extracted from plasma, separated and quantified on a Chiral-AGP column using ultraviolet detection. The simplicity, sensitivity and precision of the method makes it convenient for use in pharmacokinetic studies.     

Tissue extraction and high-performance liquid chromatographic determination of ketoprofen enantiomers

Local transcutaneous delivery of non-steroidal anti-inflammatory drugs avoids gastrointestinal side effects and concentrates drugs in the intended tissues. An extraction and HPLC method was developed for ketoprofen in skin, fascia and muscle. Tissue samples were homogenized in NaHCO₃. After methylene chloride removal of lipids, the aqueous layer was acidified with HCl and back extracted into isooctane/isopropanol. Ketoprofen was derivatized with ethylchloroformate/S-(−)-α-phenylethylamine in triethylamine, then detected by HPLC. Ketoprofen recovery was linear (1–33 μg/g) and was detected in these tissues following in vivo cathodic iontophoresis (160 mA*min). This represents the first non-radioactive method for determination of ketoprofen in tissues following transcutaneous iontophoresis.     

High-performance liquid chromatographic determination of thiopentene enantiomers in sheep plasma

An HPLC method was developed to determine the plasma concentrations of R(+)- and S(−)-thiopentone for pharmacokinetic studies in sheep. The method required separation of the thiopentone enantiomers from the corresponding pentobarbitone enantiomers which are usually present as metabolites of thiopentone. Phenylbutazone was used as an internal standard. After acidification, the plasma samples were extracted with a mixture of ether and hexane (2:8). The solvent was evaporated to dryness and the residues were reconstituted with sodium hydroxide solution (pH 10). The samples were chromatographed on a 100 mm × 4 mm I.D.. Chiral AGP-CSP column. The mobile phase was 4.5% 2-propanol in 0.1 M phosphate buffer (pH 6.2) with a flow-rate of 0.9 ml/min. This gave k′ values of 1.92, 2.92, 5.71, 9.30 and 11.98 for R(+)-pentobarbitone, S(−)-pentobarbitone, R(+)-thiopentone, S(−)-thiopentone, and phenylbutazone, respectively. At detection wavelength of 287 nm, the limit of quantitation was 5 ng/ml for R(+)-thiopentone and 6 ng/ml for S(−)-thiopentone. The inter-day coefficients of variation at concentrations of 0.02, 0.1 and 8 μg/ml were, respectively, 4.8, 4.4 and 3.5% for R(+)-thiopentone and, respectively, 5.0, 4.3 and 3.9% for S(−)-thiopentone (n = 6 each enantiomer). At the same concentrations, the intra-day coefficients of variation from six sets of replicates (measured over six days) were, respectively, 8.0, 8.0 and 8.8% for R(+)-thiopentene and 8.8, 7.4 and 9.6% for S(−)-thiopentone. Linearity over the standard range, 0.01–40 μg/ml, was shown by correlation coefficients> 0.998. This method has proven suitable for pharmacokinetic studies of thiopentone enantiomers after administration of rac-thiopentone in human plasma also and would be suitable for pharmacokinetic studies of the pentobarbitone eantiomers.     

Development and validation of a high-performance liquid chromatographic method for the determination of methocarbamol in human plasma

An isocratic HPLC method was developed and validated for the quantitation of methocarbamol in human plasma. Methocarbamol and internal standard in 200 μl of human plasma were extracted with ethyl acetate, evaporated to dryness and reconstituted in water. Separation was achieved on a reversed-phase C₁₈ column with a mobile phase of methanol—0.1 M potassium phosphate monobasic—water (35:10:55, v/v/v). The detection was by ultraviolet at 272 nm. Linearity was established at 1–100 μg/ml (r > 0.999). The limit of quantitation was designed as 1 μg/ml to suit pharmacokinetic studies. Inter-day precision and accuracy of the calibration standards were 1.0 to 3.6% coefficients of variance (C.V.) and −2.0 to +1.6% relative error (R.E.). Quality controls of 3, 20 and 70 μg/ml showed inter-day precision and accuracy of 2.5 to 3.6% C.V. and −0.9 to −0.4% R.E. Recovery of methocarbamol was 91.4–100.3% in five different lots of plasma. The method was shown to be applicable on different brands of C₁₈ columns.     

Simultaneous quantitative determination of the major phase I and II metabolites of ibuprofen in biological fluids by high-performance liquid chromatography on dynamically modified silica

Ibuprofen has previously, after ingestion by man, been demonstrated to yield four major phase I metabolites, which are excreted in the urine partly as glucuronic acid conjugates. However, in previous investigations the quantitative determinations of the conjugates were performed by indirect methods. The purpose of the present investigation was to develop a high-performance liquid chromatographic (HPLC) system for the simultaneous determination of the major phase I and II metabolites of ibuprofen in biological fluids. The separation was performed using bare silica dynamically modified with N-cetyl-N,N,N-trimethylammonium hydroxide ions contained in the mobile phase. The separation of the metabolites of ibuprofen is greatly improved with this system compared to other published reversed-phase HPLC systems intended for the same purpose. The method developed makes it possible to simultaneously determine the intact glucuronic acid conjugates of ibuprofen as well as its phase I metabolites in human urine. In a study involving four healthy volunteers, a total recovery in urine of the dose given was found to be 58–86% within 8 h. This may be compared to an average of 67% earlier reported in the literature.