Ion-pair extraction and liquid chromatographic analysis of morphine in rat brain and plasma

A highly efficient and reproducible two-step liquid—liquid ion-pair extraction technique for the isolation of morphine from biological samples is described. A rapid normal phase high-performance liquid chromatographic procedure coupled with amperometric electrochemical detection has also been developed for subsequent quantification of morphine. Extraction involves the disruption of brain tissue or plasma in methanol, centrifugation, evaporation and reconstitution in ethyl acetate containing 10 mM di-(2-ethylhexyl) phosphoric acid, a liquid cation-exchanger, and back-extraction into 170 mM orthophosphoric acid. An acidic eluent consisting of acetonitrile—76 mM orthophosphoric acid—ammonia buffer (pH 3.0) (15:85, v/v) in combination with a strong cation-exchange column allows complete separation of morphine and the internal standard, nalbuphine. The limit of detection for morphine is 1.3 ng on-column.     

High-performance liquid chromatographic method for the quantitative determination of butorphanol, hydroxybutorphanol, and norbutorphanol in human urine using fluorescence detection

A sensitive, quantitative reversed-phase high-performance liquid chromatographic method has been established for the simultaneous determination of butorphanol, a synthetic opioid, and its metabolites, hydroxybutorphanol and norbutorphanol, in human urine samples. The method involved extraction of butorphanol, hydroxybutorphanol, and norbutorphanol from urine (1.0 ml), buffered with 0.1 ml of 1.0 M ammonium acetate (pH 6.0), onto 1-ml Cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the HPLC mobile phase, acetonitrile—methanol—water (20:10:70, v/v/v), containing 10 mM ammonium acetate and 10 mM TMAH (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5-μm column. The analysis was accomplished by detection of the fluorescence of the three analytes, at excitation and emission wavelengths of 200 nm and 325 nm, respectively. The retention times for hydroxybutorphanol, norbutorphanol, the internal standard, and butorphanol were 5.5, 9.0, 13.0, and 23.4 min respectively. The validated quantitation range of the method was 1–100 ng/ml for butorphanol and hydroxybutorphanol, and 2–200 ng/ml for norbutorphanol in urine. The observed recoveries for butorphanol, hydroxybutorphanol, and norbutorphanol were 93%, 72%, and 50%, respectively. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of study samples. The method was applied on study samples from a clinical study of butorphanol, providing a pharmacokinetic profiling of butorphanol.     

Determination of trimethoprim in bovine serum by high-performance liquid chromatography with confirmation by thermospray liquid chromatography—mass spectrometry

A high-performance liquid chromatographic (HPLC) method with a detection limit of 5 ng/ml was developed for the analysis of trimethoprim in bovine serum. Trimethoprim and the internal standard, ormetoprim, under alkaline conditions, were first extracted into dichloromethane and then back-extracted into dilute sulphuric acid (0.15 M) and cleaned-up on a C₁₈ cartridge. Trimethoprim was quantified on a C₁₈ column using a triethylammonium acetate—acetonitrile—methanol (16:3:1, v/v/v) mobile phase at a flow-rate of 1.5 ml/min, with ultraviolet detection at 225 nm. This method was used to verify the accuracy of test responses obtained with the Brilliant Black Reduction test, a rapid screening method, for trimethoprim levels in the serum of steers treated with Trivetrin. Confirmation of the presence of trimethoprim in the sample extract was obtained by thermospray HPLC—mass spectrometry.     

Simple high-performance liquid chromatographic method for the determination of PGT/1A, a new immunostimulating drug, in biological fluids

This paper describes a simple high-performance liquid chromatographic method for the determination of PGT/1A (3- -pyroglutamyl- -thiazolidine-4-carboxylic acid), a new immunostimulating drug, in plasma and urine. The column was packed with LiChrospher-NH₂ (5 μm), the mobile phase was 0.02 M monobasic potassium phosphate (pH 3.2 with concentrated phosphoric acid)—acetonitrile (25:75, v/v), the flow-rate was 1.2 ml/min, the detection wavelength was 210 nm and the apparatus was a Varian Model 5000. Plasma (1 ml) was added to 1.2 ml of acetonitrile and the supernatant injected; the urine was diluted 1:5. The retention time of PGT/1A was 9.4 min in plasma and 9.9 min in urine. The method was validated for recovery, accuracy and reproducibility. The results after intravenous injection in twelve volunteers are also given.     

Determination of azosemide and its metabolite in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the determination of azosemide and its metabolite, M1, in human plasma and urine and rabbit blood and tissue homogenates. The methods involved deproteinization of the biological samples: 2.5 volumes of acetonitrile were used for the determination of azosemide and 1 volume of saturated Ba(OH)₂ and ZnSO₄ for that of M1. A 50-μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column in each instance. The mobile phases employed were 0.03 M phosphoric acid—acetonitrile (50:40, v/v) for azosemide and 0.03 M phosphoric acid/0.2 M acetic acid—acetonitrile (83:17, v/v) for M1. The flow-rate was 1.5 ml/min in both instances. The column effluent was monitored by ultraviolet detection at 240 and 236 nm for azosemide and M1, respectively. The retention times for azosemide and M1 were 6.0 and 8.3 min, respectively. The detection limits for both azosemide and M1 in both human plasma and urine were 50 ng/ml. The coefficients of variation of the assay were generally low (below 11.0%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or other diuretics tested were observed.     

Detection and separation of the anthrapyrazole CI-941 and its metabolites in serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method using ion-pairing chromatography on reversed-phase C₁₈ material with a mobile phase of acetonitrile—water (19:81, v/v) containing 5 mM 1-pentanesulfonic acid was developed for the detection and separation of the anthrapyrazole CI-941 (I) and its metabolites. After sample clean-up with solid-phase extraction, I and its metabolites were measurable at a wavelength of 491 nm. A detection limit of 5 ng/ml was achievable for I. The dicarboxylic acid derivative and the isomers of the monocarboxylic acid derivative could be separated. Application of the method to a human pharmacokinetic study showed two and four metabolites of I in serum and urine respectively.     

Determination of a novel sigma receptor antagonist, DuP 734, in rat plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A selective high-performance liquid chromatographic (HPLC) assay for a sigma receptor antagonist, DuP 734 (I), in rat plasma has been developed. Compound I and internal standard, XC031 (I.S.), were first extracted from plasma into an ethyl acetate—toluene mixture (3:7, v/v) and then back-extracted into freshly prepared phosphoric acid (0.03 M). Separation of I and I.S. with no interference from endogenous substances was achieved on a reversed-phase octyl column and detection was by UV at 229 nm. The mobile phase consisted of acetonitrile—glacial acetic acid—triethylamine—0.05 M ammonium acetate (670:4:2:2000, v/v). Using 0.5 ml of rat plasma for extraction, the limit of quantitation was 43 ng/ml and the assay was linear from 43 to 8536 ng/ml. The intra- and inter-day coefficients of variation ranged from 0.7 to 3.0%, and from 1.4 to 14.5%, respectively, over the entire concentration range. The accuracy was within 16.1% of the spiked concentrations. I was stable in frozen plasma at −20°C for at least 68 days.     

Quantification of total and free mycophenolic acid in human plasma by liquid chromatography with fluorescence detection

A simple high-performance liquid chromatographic (HPLC) method was developed for the assay of total and free mycophenolic acid (MPA) in human plasma. Prior to analysis, total mycophenolic acid was extracted by protein precipitation and free drug was isolated from plasma samples using ultrafiltration. The extracts were injected onto a Kromasil C8 column at 30 degrees C with excitation and emission wavelengths set at 342 and 425 nm, respectively. The mobile phase was consisted of acetonitrile-32 mM glycine buffer, pH 9.2 (20:80, v/v), at a flow rate of 1.0 ml/min. The method was found to be linear over the concentration range investigated, 0.05-40 mg/l for total mycophenolic acid (r>0.999) and 5-1000 microg/l (r>0.99) for free drug. The percentage error of the analytical method was below 10.9%. The intra- and inter-day reproducibility was adequate with the coefficients of variation of 8.28% or below. The run time were 4 and 6 min for free and total MPA, respectively. The method thus can be effectively applied to measure mycophenolic acid concentrations in clinical samples.     

Determination of timiperone in rat plasma by high-performance liquid chromatography with electrochemical detection

We report a sensitive new method for the determination of timiperone in rat plasma by using high-performance liquid chromatography with electrochemical detection. The method involves extraction of plasma samples with heptane-isoamyl alcohol at pH>8, followed by back-extraction into dilute acetic acid. Separation was accomplished by reversed-phase high-performance liquid chromatography on an ODS column with the mobile phase consisting of 0.1 M phosphate buffer (pH 3.5)-acetonitrile-methanol (65:20:15, v/v). Recovery was greater than 80%. Calibration curve was linear over the concentration range 0.5–50.0 ng/ml. The limit of quantitation of timiperone was 0.5 ng/ml plasma.     

Determination of rhoifolin and daidzin in human plasma by high-performance liquid chromatography

A method for determining flavonoids in human plasma is presented for application to pharmacokinetic studies of two flavonoids, rhoifolin and daidzin. Isocratic reversed-phase high-performance liquid chromatography (HPLC) was used with genistin as an internal standard and solid-phase extraction using a Sep-Pak C₁₈ cartridge. The mobile phases were acetonitrile—0.1 M ammonium acetate solution (20:80, v/v) for rhoifolin and methanol—0.1 M ammonium acetate solution (33:67, v/v) for daidzin. The detection limits on-column were 2 ng for rhoifolin and 0.5 ng for daidzin.     

Determination of picotamide in human plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of picotamide in human plasma and urine is described. After addition of an internal standard (bamifylline), the plasma and urine samples were subjected to liquid—liquid extraction and clean-up procedures. The final extracts were evaporated to dryness and the resulting residues were reconstituted in 100 μl of methanol—water (50:50, v/v) and chromatographed on a LiChrosorb RP-SELECT B reversed-phase column coupled to an ultraviolet detector monitored at 230 nm. Chromatographic analysis takes about 10 min per sample. The assay was linear over a wide range and has a limit of detection of 0.005 and 0.1 μg/ml in plasma and urine, respectively. It was selective for picotamide, accurate and robust and thus suitable for routine assays after therapeutic doses of picotamide.     

High-performance liquid chromatographic determination of naproxen, ibuprofen and diclofenac in plasma and synovial fluid in man

High-performance liquid chromatographic assay procedures have been developed for naproxen, ibuprofen and diclofenac in human plasma and synovial fluid samples. A single liquid—liquid extraction procedure was used to isolate each compound from acidified biological matrix prior to the quantitative analysis. A Spherisorb ODS column (12.5 cm × 4.6 mm I.D.) was used for all the chromatography. Naproxen was eluted with a mobile phase of methanol—Sörensen's buffer at pH 7 (37:63, v/v). Ibuprofen and diclofenac were eluted using mobile phases of methanol—water at pH 3.3 (65:35, v/v and 63:37, v/v, respectively). Diphenylacetic acid was used as the internal standard for the assay of naproxen and flurbiprofen was used in the analysis of ibuprofen and diclofenac. Inter- and intra-day coefficients of variation were less than 7%. The assays were used in clinical studies of the three drugs in osteo- and rheumatoid arthritis patients.     

Liquid chromatographic method for the simultaneous determination of caffeine and fourteen caffeine metabolites in urine

An HPLC method has been developed for the separation and the determination of caffeine and its metabolites in urine samples using a one extraction–analysis run and UV detection. The compounds were extracted by liquid–liquid extraction using chloroform–isopropylalcohol (85:15, v/v). Chromatographic separation was accomplished on an ODS analytical column with a mobile phase containing 0.05% acetic acid/methylalcohol (92.5:7.5, v/v). Compounds were monitored at 280 nm. The method was validated for the determination of AFMU, 1X, 1U, 17X and 17U caffeine metabolites required to assess the metabolic activity of the enzymes subject to in vivo caffeine testing. The validated assay was applied to urine samples from ten healthy volunteers. The method was proved to be suitable to assess simultaneously the enzymatic activity of cytochrome P450 CYP1A2 and CYP2A6, as well as N-acetyltransferase and xanthine oxidase.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.     

High-performance liquid chromatographic determination of sotalol in biological fluids

A sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of sotalol in human serum and urine. Sotalol and the internal standard, atenolol, were extracted from alkalinized serum and urine (pH 9.0) into 1-butanol—chloroform (20:60, v/v). The organic phase was evaporated, and to the residue was added 0.1 M sulphuric acid (serum analysis) or mobile phase (urie analysis). The mobile phase consisted of 0.01 M phosphate buffer (pH 3.2) and acetonitrile (20:80, v/v) containing 3 mM n-octylsodium sulphate. The flow-rate was 1.5 ml/min. The retention times of atenolol and sotalol were 7 and 10 min, respectively. Ultraviolet detection at 226 nm made it possible to achieve a detection limit of 0.03 μmol/l.     

Determination of a new antifilarial drug, UMF-058, and mebendazole in whole blood by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for simultaneous quantitative determination of a new antifilarial drug (UMF-058, I) and mebendazole (MBZ) is described. After a simple extraction from whole blood, both compounds were analysed using a C₁₈ Nova Pak reversed-phase column and a mobile phase of methanol—0.05 M ammonium dihydrogenphosphate (50:50, v/v) adjusted to pH 4.0, with ultraviolet detection at 291 nm. The average recoveries of I and MBZ over a concentration range of 25–250 ng/ml were 92.0 ± 7.7 and 84.4 ± 4.4%, respectively. The minimum detectable concentrations in whole blood for I and MBZ were 7 and 6 ng/ml, respectively. This method was found to be suitable for pharmacokinetic studies.     

Stereoselective Determination of a Novel Chiral Insecticide,Sulfoxaflor, in Brown Rice,Cucumber and Apple by Normal‐Phase High‐Performance Liquid Chromatography

An effective high‐performance liquid chromatography method was developed for the stereoselective determination of a new sulfoximines insecticide, sulfoxaflor, in brown rice, cucumber and apple. Target compounds were extracted with acetonitrile and an aliquot cleaned with Cleanert PestiCarb/PSA (primary and secondary amine) cartridge. Five polysaccharide‐based columns were investigated on the separation of sulfoxaflor stereoisomers and the best was achieved on a ChromegaChiral CCA column with n‐hexane/ethanol/methanol (90:2:8, v/v/v) as mobile phase by UV detection at 220 nm at 20ºC. The resolutions of the four stereoisomers were 1.85, 1.54 and 3.08, and the elution order was identified by optical rotation and stereoisomers ratio. The mean recoveries of sulfoxaflor stereoisomers ranged from 77.1% to 99.3%, with relative standard deviations less than 8.9% at three concentration levels in all matrices. The limits of detection for all stereoisomers varied from 0.05 mg/kg to 0.07 mg/kg, while the limit of quantification did not exceed 0.22 mg/kg. The method was then successfully applied to determine the sulfoxaflor stereoisomers in authentic samples, confirming that it is convenient and reliable for stereoselective determination of sulfoxaflor stereoisomers in food. Chirality 26:114–120, 2014. © 2014 Wiley Periodicals, Inc.     

Simultaneous determination of dopamine, dopac and homovanillic acid : Direct injection of supernatants from brain tissue homogenates in a liquid chromatography—electrochemical detection system

A simple method based on high-performance liquid column chromatography with electrochemical detection is described for the simultaneous determination of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in discrete brain regions of rats. The supernatant of a tissue homogenate is injected directly onto a liquid chromatograph, thus omitting the commonly adopted adsorption step. Of the four different supports tested Nucleosil C₁₉ (5 μm) was found superior with respect to chromatographic performance. The effects of pH, methanol and the ion-pairing agent hexyl sulfate on the retention were studied. The mobile phase used in the final studies consisted of citrate buffer pH 4.25—methanol (92:8, v/v) containing hexyl sulfate (1.7 · 10⁻³M). Standard curves of dopamine, DOPAC and HVA were found linear up to about 600 pmol per injection for each compound. The precisions of the chromatographic step were (s_rel. %): 0.72% (dopamine), 1.26% (DOPAC) and 2.69% (HVA).     

Liquid chromatographic determination of urinary 2-thiothiazolidine-4-carboxylic acid, a biomarker of carbon disulphide exposure

An effective gradient high-performance liquid chromatographic method for baseline separation of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), with photodiode array detection at 271 nm was described. o-Methylhippuric acid was used as an internal standard (I.S.). A 1-ml urine sample was saturated with 300 mg of sodium sulphate, acidified with 100 μl of 6 M hydrochloric acid, extracted twice with 2 ml of diethyl ether, and after evaporation, the residue was taken up in 1 ml of 0.1% (v/v) phosphoric acid. The two mobile phases used for gradient elution were: (A) 10 mM ammonium dihydrogenphosphate (pH 3.5) and (B) same concentration of buffer but containing 20% (v/v) of methanol (pH 4.8). The flow-rate was set at 1.0 ml/min. TTCA and I.S. were detected at 2.2 and 9.1 min, respectively. The method was validated with urine samples collected from normal subjects and workers occupationally exposed to carbon disulphide. The present method enables the detection of urinary TTCA at a concentration of 0.025 mg/l. Analytical recovery and reproducibility generally exceeded 90%. The proposed method is considered more sensitive, specific and reliable than other existing methods.     

Determination of a new non-narcotic analgesic, DA-5018, in plasma, urine and bile by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the determination of a new non-narcotic analgesic, DA-5018 (I), in rat plasma, urine and bile samples, using propranolol for plasma samples and protriptyline for urine and bile samples as internal standards. The method involved extraction followed by injection of 100 μl of the aqueous layer onto a C₁₈ reversed-phase column. The mobile phases were 5 mM methanesulfonic acid with 10 mM NaH₂PO₄ (pH 2.5)-acetonitrile, 70:30 (v/v) for plasma samples and 75:25 (v/v) for urine and bile samples. The flow-rates were 1.0 ml/min for plasma samples and 1.2 ml/min for urine and bile samples. The column effluent was monitored by a fluorescence detector with an excitation wavelength of 270 nm and an emission wavelength of 330 nm. The retention time for I was 4.8 min in plasma samples and 10.0 min in urine and bile samples. The detection limits for I in rat plasma, urine and bile were 20, 100 and 100 ng/ml, respectively. There was no interference from endogenous substances.