Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Solid-phase extraction and reversed-phase high-performance liquid chromatographic determination of sulphur mustard in blood

A reversed-phase high-performance liquid chromatographic method is reported for the analysis of sulphur mustard in blood with the aid of solid-phase extraction sample preparation. Sulphur mustard is extracted from blood samples (both in vitro and in vivo) of rats with a solution of 0.05 M sodium dodecyl sulphate and pre-concentrated over Sep-Pak C₁₈cartridges pre-coated with Tween-20. A Polygosil C₁₈ column is used with acetonitrile—water (52:48, v/v) as mobile phase for separation and sulphur mustard was detected at 200 nm.     

Solid-phase extraction and determination of ranitidine in human plasma by a high-performance liquid chromatographic method utilizing midbore chromatography

An improved high-performance liquid chromatographic (HPLC) method utilizing solid-phase extraction (SPE) and midbore chromatography was developed for the determination of ranitidine in human plasma. A mobile phase of 20 mM K₂HPO₄-acetonitrile-triethylamine (87.9:12.0:0.1, v/v) pH 6.0 was used with a phenyl analytical column and ultraviolet detection (UV). The method demonstrated linearity from 25 to 1000 ng/ml in 500 μl of plasma with a detection limit of 10 ng/ml. The method was utilized in a pharmacokinetic study evaluating the effects of pancreatico-biliary secretions on ranitidine absorption.     

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.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

Novel high-performance liquid chromatographic and solid-phase extraction methods for quantitating methadone and its metabolite in spiked human urine

A novel solid-phase extraction (SPE) method and HPLC method were developed for the determination of methadone and its metabolite from spiked human urine. For sample cleanup, a spiked urine sample was pretreated with phosphoric acid followed by a well-thought-out SPE method using a 10-mg Oasis HLB 96-well extraction plate. In this SPE method, the concentration of methanol as well as the pH are optimized to preferentially isolate the analytes of interest from the sample matrix. Low elution volumes (200 μl) are achieved; this eliminates evaporation and reconstitution of the sample solution. Recoveries from human urine matrix were greater than 91% with RSD values less than 4.5%. For the HPLC analysis, the separation was obtained using a SymmetryShield RP₁₈ column with a mobile phase of 0.1% TFA–methanol (60:40, v/v). Good peak shapes were obtained without the need of addition of any competing reagent to the mobile phase. Additionally, significant signal-to-noise enrichment was achieved by diluting the final SPE eluates four-fold with water.     

Reliable and specific high-performance liquid chromatographic method for simultaneous determination of loratadine and its metabolite in human plasma

A high-performance liquid chromatographic (HPLC) method with fluorescence detection has been developed for the simultaneous determination of loratadine (L) and its metabolite, descarboethoxyloratadine (DCL), in human plasma. Following a two-step liquid-liquid extraction with toluene, the analytes were separated using a gradient mobile phase consisting of methanol-acetonitrile-phosphate buffer. The linearity for L and DCL was within the concentration range of 0.5-16 ng/ml. The coefficient of variation of intra- and inter-day assay was <8.3%, with accuracy ranging from 98.3 to 105.7%. The lower limit of quantification was 0.5 ng/ml for both L and DCL. This method has been demonstrated to be reliable, and is an improvement over existing methods due to its capability for determining L and DCL simultaneously in a single chromatographic run.     

Rapid high-performance liquid chromatographic determination of serum gabapentin

Gabapentin (GBP) is a new antiepileptic drug approved for clinical treatment of partial seizures in the USA. Serum GBP concentrations in 283 patients were studied using high-performance liquid chromatography with fluorescence detection. The standard curves were linear over a range of 60 ng to 15 μg/ml. The coefficient of variations were 3.4 to 8.8% and 1.4 to 9.8% for intra- and inter-assay studies, respectively. The lower limit of quantitation was 10 ng/ml. Of the 283 patients studied, 72.5% had GBP levels between 2 and 10 μg/ml, 14.8% were below 2 μg/ml and 12.7% above 10 μg/ml. The mean±S.E. of GBP in 283 patients was 5.38±0.23 μg/ml. Peak concentrations of more than 15 μg/ml and trough levels as low as 0.1 μg/ml were not uncommon. The method described was rapid, simple, highly sensitive and reproducible. Other antiepileptic drugs and endogenous compounds did not interfere with the assay.     

Simplified, rapid and inexpensive extraction procedure for a high-performance liquid chromatographic method for determination of disopyramide and its main metabolite mono-N-dealkylated disopyramide in serum

A simplified, rapid and inexpensive extraction procedure for the determination of the antiarrhythmic drug disopyramide and its main metabolite mono-N-desalkylated disopyramide in serum by high-performance liquid chromatography has been developed. The analysis uses ultraviolet detection at 254 nm, and a 5 μm reversed-phase column with a mobile phase of water—triethylamine—acetonitrile—PIC-B8 reagent. Serum extraction is performed with dichloromethane and 1 M sodium hydroxide. p-Chlorodisopyramide is used as internal standard. Recovery rates were 94.5% (S.D. 5.7%) for disopyramide, 96.8% (S.D. 2.2%) for mono-N-desalkylated disopyramide and 97.9% (S.D. 2.8%) for the internal standard.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical 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.     

Column liquid chromatographic determination of paroxetine in human serum using solid-phase extraction

A 0.5-ml aliquot of a serum sample, after the addition of a 100-μl aliquot of a 5 μg/ml solution of dibucaine as the internal standard, is vortex-mixed with 0.5 ml of acetonitrile and centrifuged. The supernatant is applied to a 1-ml BondElut C₁₈ silica extraction column conditioned with subsequent washings with 1 M HCl, methanol and water. After passing the sample at a slow rate, the column is washed twice with water and once with acetonitrile. The desired compounds are then eluted with a 0.25-ml aliquot of 35% perchloric acid—methanol (1:40, v/v). A 7-μl aliquot of the eluate is injected onto a 150 × 4.6 mm I.D. column packed with 5-μm C₈ silica particles and eluted at ambient temperature with a mobile phase of 10 mM phosphate buffer-acetonitrile (2:1, v/v) (pH 3.2). The peaks are detected with a fluorescence detector (excitation at 295 nm, emission at 365 nm). The resulting chromatogram is clean with no extraneous peaks. Paroxetine and dibucaine give sharp peaks which are well separated from each other and from the solvent peaks. The extraction recovery of the drug and the internal standard is in the range of 90% which allows a highly sensitive determination of paroxetine.     

Improved high-performance liquid chromatographic determination of ciprofloxacin and its metabolites in human specimens

A simple approach to the quantitation of ciprofloxacin and its three metabolites, M1 (desethylene-ciprofloxacin), M2 (sulfo-ciprofloxacin) and M3 (oxo-ciprofloxacin), in human serum, urine, saliva and sputum is described. This assay allows the parent drug and its metabolites to elute and be resolved in a single chromatogram at 280 nm using a linear gradient. The procedure involved liquid—liquid extraction. Separation was achieved on a C₁₈ reversed-phase column. The limit of detection of ciprofloxacin is 0.05 μg/ml and that of its three metabolites is 0.25 μg/ml. This method is sufficiently sensitive for pharmacokinetic studies.     

Simultaneous stereoselective high-performance liquid chromatographic determination of 10-hydroxycarbazepine and its metabolite carbamazepine-10,11-trans-dihydrodiol in human urine

An enantioselective HPLC method for the simultaneous determination of the concentration of the enantiomers of the oxcarbazepine metabolites 10-hydroxycarbazepine (MHD) and carbamazepine-10,11-trans-dihydrodiol (DHD) in human urine is described. The method is based on extraction with tert.-butylmethyl ether–dichloromethane (2:1, v/v) under alkaline conditions, separation and evaporation of the organic phase and dissolution of the residue in the mobile phase. Enantiomers are resolved on a Diacel Chiralcel OD column (250 mm×4.6 mm I.D.) under isocratic conditions using as mobile phase n-hexane–ethanol–2-propanol (18:2:1, v/v/v) with addition of glacial acetic acid (0.1%). The enantiomers are detected by UV at 215 nm. The method allows reliable determination of the MHD and DHD enantiomers in human urine with limits of quantification of 0.2 mg/l and 0.4 mg/l, respectively.     

Double column-switching high-performance liquid chromatographic method for the determination of TAK-603 and its metabolites in human serum

A double column-switching high-performance liquid chromatographic (HPLC) method for the determination of concentrations for TAK-603 (T) and its metabolites, T-72258 (M-I) and T-72294 (M-III), in human serum was developed. The analytes were extracted with ethyl acetate from human serum samples treated with triethylamine and injected into the HPLC system. Separation of the analytes was performed on the HPLC system with double column-switching technique. The mobile phases A and B for the first column and the mobile phase C for the second column used were a mixture of methanol–10 mM aqueous ammonium acetate solution (1:1, v/v), methanol and a mixture of methanol–10 mM aqueous ammonium acetate solution (11:9, v/v), respectively. The eluate was monitored with a UV detector at a wavelength of 253 nm. The work-up procedure was reproducible and more than 90% of the analytes could be recovered from human serum. The lower limits of quantitation were all 1 ng/ml for the analytes when 0.5 ml of human serum was used. Standard curves were linear with a correlation coefficient (R) of more than 0.999 in the range of 1–500 ng/ml for T, M-I and M-III in human serum. The intra- and inter-day precision of the method for the various analytes were below 4.8%. The accuracy was good with the deviations between spiked and calculated concentrations of the analytes being within 11.0%. The method was successfully applied to analyze serum samples after an oral administration of T to healthy male volunteers.     

Reversed-phase liquid chromatographic determination of idarubicin and its 13-hydroxy metabolite in human plasma

A method is given for the determination of idarubicin and its main metabolite, idarubicinol, in plasma from cancer patients. Idarubicin and idarubicinol are extracted from 2-ml samples of buffered plasma (pH 8.1) using chloroform-1-heptanol (9:1). After reextraction into phosphoric acid (0.1 M), separation is performed by reversed-phase liquid chromatography on a LiChrosorb RP-2 column (5 μm) with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The absolute recovery in the range 5–100 ng/ml was greater than 83% with a precision better than 8% (relative standard deviation), using photometric detection at 484 nm. Proper handling of whole blood samples containing idarubidin is essential to avoid metabolic conversion into idarubicinol. Prolonged storage of the drug and its main metabolite under alkaline conditions should be avoided to prevent chemical degradation.     

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.     

On-line solid-phase extraction and high-performance liquid chromatographic determination of chlorthalidone in urine

A simple and rapid on-line method for the determination of chlorthalidone in urine is proposed. The sample containing the internal standard is injected in a CN precolumn. After a 2-ml water rinsing, the precolumn is coupled for 30 s to the HPLC column via a switching valve, allowing the on-line elution of the compounds of interest. Analysis is carried out by reversed-phase chromatography with an acetonitrile-0.01 M phosphate buffer pH 7 (20:80, v/v) eluent, using UV detection at 214 nm. While the LC separation is performed, the precolumn is regenerated and conditioned, and is ready to receive the next sample at the end of the run. Accurate (>95%) and precise (<10%) analyses, in the range of 0.1–20 μg/ml of chlorthalidone in urine, have been achieved using this method.     

A validated high-performance liquid chromatographic assay for the simultaneous determination of denaverine and its N-monodemethyl metabolite in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of denaverine and its N-monodemethyl metabolite (MD 6) in human plasma is described. The assay involves the extraction with an n-heptane–2-propanol mixture (9:1, v/v) followed by back extraction into 12.5% (w/w) phosphoric acid. The analytes of interest and the internal standard were separated on a Superspher RP8 column using a mobile phase of acetonitrile–0.12 M NH₄H₂PO₄–tetrahydrofuran (24:17.2:1, v/v), adjusted to pH 3 with 85% (w/w) phosphoric acid. Ultraviolet detection was used at an operational wavelength of 220 nm. The retention times of MD 6, denaverine and the internal standard were 5.1, 6.3 and 10.2 min, respectively. The assay was validated according to international requirements and was found to be specific, accurate and precise with a linear range of 2.5–150 ng/ml for denaverine and MD 6. Extraction recoveries for denaverine and MD 6 ranged from 44 to 49% and from 42 to 47%, respectively. The stability of denaverine and MD 6 in plasma was demonstrated after 24 h storage at room temperature, after three freeze–thaw cycles and after 7 months frozen storage below −20°C. The stability of processed samples in the autosampler at room temperature was confirmed after 24 h storage. The analytical method has been applied to analyses of plasma samples from a pharmacokinetic study in man.