Rapid determination of chloroprocaine and its major metabolite, 2-chloroaminobenzoic acid, in plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic method for determination of the 2-chloroprocaine, local anesthetic of ester type, and its major metabolite 2-chloroaminobenzoic acid, has been developed and validated. A single-step extraction procedure is employed followed by high-performance liquid chromatographic separation using reversed-phase column and analysis using variable length UV detection. Lidocaine was used as internal standard for 2-chloroprocaine measurement and p-aminobenzoic acid was used as internal standard for 2-chloroaminobenzoic acid analysis. The analysis of spiked plasma demonstrated good accuracy and precision of the method with limit of detection 0.1 μg/ml for 2-chloroprocaine and 0.5 μg/ml for 2-chloroaminobenzoic acid. The method has been used for pharmacokinetic studies in laboratory animals.     

Rapid determination of PEGylated liposomal doxorubicin and its major metabolite in human plasma by ultraviolet-visible high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of doxorubicin derived from PEGylated liposomal doxorubicin (Doxil) and its major metabolite in human plasma. This method utilizes Triton X-100 to disperse the liposome, followed by a protein precipitation step with 5-sulfosalicylic acid. Analytes in the resultant supernatant are separated on a Discovery RP amide C(16) column (250 x 3 mm I.D., 5 microm) using an isocratic elution with a mobile phase consisting of 0.05 M sodium acetate (pH 4.0) and acetonitrile (72:28). The retention times for doxorubicin and the internal standard daunorubicin were 4.8 and 10.1 min, respectively. The column eluate was monitored by UV-visible detection at 487 nm. The determination of doxorubicin was found to be linear in the range of 1.0 ng/mL to 25 microg/mL, with intra-day and inter-day coefficients of variation and percent error < or =10%. The recovery of doxorubicin from plasma was >69.3%, with a liposomal dispersion efficiency of >95.7%. Our analytical method for free and PEGylated doxorubicin in human plasma is rapid, avoids organic extractions, and maintains sensitivity for the parent compound and its major metabolite, doxorubicinol.     

Rapid and simple method for the determination of zolpidem in human plasma by high-performance liquid chromatography

A simple and reproducible method for the determination of zolpidem in human plasma is presented. This method involves protein precipitation with methanol (2 ml of methanol are added to 0.5 ml of plasma) and reversed-phase chromatography with fluorescence detection (excitation wavelength 244 nm, emission wavelength 388 nm). The mobile phase consists of methanol–30 mM dihydrogen potassium phosphate–triethylamine (30:69:1). pH of the aqueous part of the mobile phase is 6.8. No internal standard is required. Limit of quantitation is 1.5 ng/ml and the calibration curve is linear up to 400 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy also does not exceed 9%. The assay is useful for pharmacokinetic studies.     

Evaluation of a modified high-performance liquid chromatography assay for acebutolol and its major metabolite

Extensive modification of an existing high-performance liquid chromatography assay for acebutolol and its major metabolite has markedly improved chromatographic stability eliminating the previous need for frequent adjustment of the eluent composition to accommodate continuous loss of column retention. The eluents now used and avoidance of the requirement for elevated column temperature may be significant factors in the ability to maintain column life over 8 months of continuous use with little decrease in retention As a result of the improved chromatographic stability full advantage can now be taken of automatic injection devices for the unattended processing of large numbers of samples. A significant modification of the work-up of blood samples has improved precision of the assay in whole blood. Nevertheless, it is recommended that plasma samples rather than whole blood be analyzed, since the plasma assay is faster and still more precise.     

Rapid method for determination of riboflavin in urine by high-performance liquid chromatography

A simple, specific, and sensitive high-performance liquid chromatographic (HPLC) method for the determination of riboflavin directly in urine samples using a fixed-wave-length spectrofluorometer is described. Centrifuged raw urine samples (50 μl) are injected onto a reversed-phase microparticulate C₁₈ column. The eluent is 0.01 M KH₂PO₄ (pH 5.0)—methanol (65:35). This method is capable of differentiating riboflavin from riboflavin-5-phosphate, non-riboflavin fluorescing components in urine, and photo-degraded riboflavin. The method shows good reproducibility and is linear to at least 12 μg/ml. The sensitivity of this procedure, at the 95% confidence limit, determined by linear regression analysis, is estimated to be 0.05 μg/ml using peak height and 0.07 μg/ml using peak area. This HPLC method is compared to an automated fluorometric method for riboflavin. The coefficient of linear regression of this comparison is Y = 0.858 + 0.893X, where X is the HPLC method and Y is the fluorometric method.     

Simultaneous determination of amiodarone and its major metabolite desethylamiodarone in plasma, urine and tissues by high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatographic method for the simultaneous assay of amiodarone and desethylarniodarone in plasma, urine and tissues has been developed. The method for plasma samples and tissue samples after homogenizing with 50% ethanol, involves deproteinization with acetonitrile containing the internal standard followed by centrifugation and direct injection of the supernatant into the liquid chromatograph. The method for urine specimens includes extraction with a diisopropyl ether—acetonitrile (95:5, v/v) mixture at pH 7.0 using disposable Clin-Elut 1003 columns, followed by evaporation of the eluate, reconstitution of the residue in methanol—acetonitrile (1:2, v/v) mixture and injection into the chromatograph. Separation was obtained using a Radial-Pak C₁₈ column operating in combination with a radial compression separation unit and a methanol–25% ammonia (99.3:0.7, v/v) mobile phase. A wavelength of 242 nm was used to monitor amiodarone, desethylamiodarone and the internal standard. The influence of the ammonia concentration in the mobile phase on the capacity factors of amiodarone, desethylamiodarone and two other potential metabolites, monoiodoamiodarone (L6355) and desiodoamiodarone (L3937) were investigated. Endogenous substances or a variety of drugs concomitantly used in amiodarone therapy did not interfere with the assay.The limit of sensitivity of the assay was 0.025 μg/ml with a precision of ± 17%. The inter- and intra-day coefficient of variation for replicate analyses of spiked plasma samples was less than 6%. This method has been demonstrated to be suitable for pharmacokinetic and metabolism studies of amiodarone in man.     

Simultaneous determination of tramadol and its major active metabolite O-demethyltramadol by high-performance liquid chromatography with electrochemical detection

A novel, highly sensitive method was developed for simultaneous determination of tramadol and its main active metabolite O-demethyltramadol (ODMT) in rat plasma. The method involves a single-step extraction procedure and a specific determination by high-performance liquid chromatography with electrochemical detection, using an ethoxy analogue of tramadol (L-233) as internal standard. The dual-electrode detector was operated in the oxidation-screening mode. Absolute recoveries of tramadol and ODMT were about 80%. Calibration curves were linear over a concentration range of 10–1000 ng/ml for ODMT and 10–10 000 ng/ml for tramadol with intra- and inter-day coefficients of variation not exceeding 10% and 15%, respectively. The limit of quantification for tramadol and ODMT was lower than 15 ng/ml and 10 ng/ml using 100 μl of plasma, respectively. The described method allows an adequate characterization of the plasma vs. time profiles for both compounds.     

Enantioselective determination of pazinaclone,a new isoindoline anxiolytic,and its active metabolite in rat plasma by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic method has been developed for the simultaneous determination of the enantiomers of pazinaclone (DN-2327), a new anxiolytic agent, and those of its active metabolite, M-II, in rat plasma. Organic solvent extraction of pazinaclone, M-II, and internal standard (I.S.) in plasma was followed by separation of the analytes from other metabolites using an achiral reversed-phase column. Fluorescence detection was employed with excitation and emission wavelengths of 328 and 367 nm, respectively. Separation of all the enantiomers and I.S. was then accomplished with normal- and chiral-phase columns connected in series. For each analyte, the lower quantitation limit was 0.5 ng/ml. The assay has been applied to a chiral inversion study in rats. Chiral conversion from one enantiomer of pazinaclone to the other hardly occurred. This method is suitable for enantioselective pharmacokinetic and toxicokinetic studies in animals.     

Enantioselective high-performance liquid chromatography determination of methadone enantiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded phase

The simultaneous determination of methadone (Mtd) enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in human urine and serum by enantioselective HPLC using a new Cyclobond I-2000 RSP column is described. After alkaline extraction from urine or serum with estazolam as an internal standard, Mtd enantiomers and its metabolite (EDDP) are separated on the previous column with reversed-mobile phase and detected at 210 nm. Peak resolutions are about 2.0 for Mtd enantiomers. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 0.5 and 4.5%. Most drugs of abuse are shown not to interfere with this technique. The method has been applied to study levels of each Mtd enantiomer and of its racemic metabolite in urine and serum of patients under maintenance treatment for opiate dependence. In urine, R-(−)-Mtd levels are always higher (about 2±0.5-fold_ than those of S-(+)-Mtd and in most cases, metabolite concentrations are greater than those of global Mtd enantiomers. However, the R-(−) enantiomer levels of residual drug in serum of some patients were lower than those of its antipode. This method is suitable for pharmacokinetic and toxicological studies of Mtd enantiomers and its major metabolite in biological fluids.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Simultaneous determination of gemcitabine and its metabolite in human plasma by high-performance liquid chromatography

Gemcitabine (dFdC) is a pyrimidine antimetabolite with broad spectrum activity against tumors. In this paper, a normal-phase high-performance liquid chromatographic method was developed for the determination of the parent drug (dFdC) and its metabolite (dFdU) in human plasma. The described sample preparation procedure for determination of dFdC and dFdU is rapid, sensitive, reproducible and simple. The linear regression equations obtained by least square regression method, were area under the curve=0.0371 concentration (ng ml(-1))+192.53 and 1.05.10(-4) concentration (ng ml(-1))-1.2693 for dFdC and dFdU, respectively. The assay for dFdC and dFdU described in the present report has been applied to plasma samples from a bladder cancer patient.     

Rapid method for trehalulose production and its purification by preparative high-performance liquid chromatography

Trehalulose was produced with a good yield by enzymatic conversion of sucrose and easily purified by preparative HPLC using a single Ca2+-based column. In addition, the structure of this sugar was confirmed by 13C and 1H n.m.r studies.     

Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum

A simple, rapid and sensitive high-performance liquid chromatographic procedure has been developed for the determination of ketamine and dehydronorketamine in equine serum. Sample preparation consisted of mixing equal volumes of serum and acetonitrile—phosphoric acid (85%)—water (20:2:78, v/v/v), followed by ultrafiltration through a 10 000 molecular mass cut-off filter. Separation of these two analytes in the ultrafiltrate was accomplished on a reversed-phase phenyl column eluted with methanol—acetonitrile—phosphate buffer solution. Ketamine and dehydronorketamine were detected by a variable photometric UV-Vis detector set at 215 nm, and confirmed by a photodiode array detector operated in the 200–320 nm range. The limit of detection for ketamine was 5–15 ng/ml in equine serum. Additionally, the dehydronorketamine peak identity was tentatively confirmed by thermospray liquid chromatography—mass spectrometry.     

Sensitive method for the determination of chloroquine and its metabolite desethyl-chloroquine in human plasma and urine by high-performance liquid chromatography

A method has been developed for the rapid quantitative analysis of chloroquine and its metabolite desethyl-chloroquine in plasma, blood and urine using high-performance liquid chromatography. An ethylene dichloride extract of the alkalinized biological samples was extracted with dilute acid and chromatographed on a reversed-phase column. Phosphate buffer in acetonitrile was used as the mobile phase with perchlorate as the counter-ion. Ultraviolet absorption at 254 or 340 nm or fluorescence detection was used. The fluorescence spectra and the fluorescence quantum yield of the substances were determined.Chloroquine and desethyl-chloroquine concentrations in the range of 10 nmol/l (UV-detection) and of 0.5 nmol/l (fluorescence detection) could be accurately measured with a relative standard deviation of 12%. The method should be adequate for therapeutic and pharmacokinetic studies.     

Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1^®) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was <10% at each of five methotrexate concentrations in the range 2.5–50 ng/ml. The limits of quantitation of methotrexate were 1 and 2.5 ng/ml for methotrexate and 7-hydroxymethotrexate, respectively (using 1 ml plasma). A robust HPLC method has been developed for the reproducible quantitation of methotrexate in plasma of patients taking a weekly dose of methotrexate for rheumatoid arthritis.     

Evaluation of an on-line solid-phase extraction method for determination of almokalant, an antiarrhythmic drug, by liquid chromatography

A fully automated liquid chromatographic method based on a Prospekt solid-phase extraction unit is described for determination of the antiarrhythmic drug almokalant in plasma. The assay comprises solid-phase extraction on a C₂ phase and separation on a C₁₈ column with fluorometric detection. In the original procedure 40 samples a day could be run unattended but by modifying the sequence in the solid-phase extraction process it was possible to increase this number to 70. The method gives an absolute recovery of 92% and a repeatability (C.V.) of 2.9% at 75 nmol/1 of plasma. The limit of quantitation is 2 nmol/1 of plasma (C.V. < 20%). As regards accuracy and precision the performance of the method is as good as the manual method based on liquid-liquid extraction. The Prospekt method is, above all, faster and requires far less manual effort.