Determination of phenylmercapturic acid in urine of benzene-exposed BDF-1 mice

This paper describes a procedure for the identification of phenylmercapturic acid in urine of benzene-exposed mice. Collected urine of benzene exposed mice was adjusted to pH 7 and applied to an anion exchanger. After extraction with diethyl ether and evaporation to dryness, the sample was dissolved in aqueous phosphoric acid and injected into the HPLC. HPLC conditions included an ODS column and an eluent consisting of tetrabutylammoniumhydrogensulfate—methanol (75:25, v/v), the absorbance wavelength was 255 nm. The detection limit of phenylmercapturic acid was 3 mg/l in mouse urine.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Determination of gentamicin in bovine milk using liquid chromatography with post-column derivatization and fluorescence detection

A method capable of separating and quantifying the three major and one minor components of gentamicin in milk has been developed. The method is capable of detecting 15 ng/ml gentamicin, based on a total of the four components. Milk samples are centrifuged at 4°C, the fat layer removed, and the samples deproteinated with 30% trichloracetic acid. After a second centrifugation, the supernatant is passed through a C₁₈ solid-phase extraction column. The column is washed with water, water-methanol (50:50) and methanol. Ammonium hydroxide (16%) in methanol is used to elute the gentamicin. The eluent is evaporated to near dryness and taken up with water. An aliquot of the sample is then mixed with an ion-pairing reagent for chromatography. Separation is achieved using pentanesulfonic acid in a water-methanol mobile phase on a C₁₈ reversed-phase column. The o-phthalaldehyde fluorescence derivatives of gentamicin are formed post-column and are detected with excitation at 340 nm and emission at 430 nm. The percent recovery of gentamicin averaged 72, 78 and 88% from milk samples fortified at 15, 30 and 60 ng/ml, respectively.     

High-performance liquid chromatographic–tandem mass spectrometric determination of 3-hydroxypropylmercapturic acid in human urine

A sensitive and specific high-performance liquid chromatographic–tandem mass spectrometric (HPLC–MS–MS) method was developed for the determination of 3-hydroxypropylmercapturic acid (3-HPMA) in human urine. Samples were extracted using ENV+ cartridges and then injected onto a C8 Superspher Select B column with acetonitrile and formic acid as eluent (5:95, v/v). N-Acetylcysteine was used as internal standard for HPLC–MS–MS. Linearity was given in the tested range of 50–5000 ng/ml urine. The limit of quantification was 50 ng/ml. Precision, as C.V., in the tested range of 50–5000 ng/ml was 1.47–6.04%. Accuracy ranged from 87 to 114%. 3-HPMA was stable in human urine at 37°C for 24 h. The method was able to quantify 3-HPMA in urine of non-smokers and smokers.     

Anion exchange SPE and liquid chromatography-tandem mass spectrometry in GHB analysis

In this study, the extraction of γ-hydroxybutyrate (GHB) from urine using solid-phase extraction (SPE) is described. SPE was performed on anion exchange columns after samples of urine had been diluted with de-ionized water. After application of the diluted samples containing GHB-d(6) as an internal standard, the sorbent was washed with deionized water and methanol and dried. The GHB was eluted from the SPE column with a solvent consisting of methanol containing 6% glacial acetic acid. The eluent was collected, evaporated to dryness, and dissolved in mobile phase (100 μL) for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in negative multiple reaction monitoring (MRM) mode. Liquid chromatography was performed in gradient mode employing a biphenyl column and a mobile phase consisting of acetontitrile (containing 0.1% formic acid) and 0.1% aqueous formic acid. The total run time for each analysis was less than 5 min. The limits of detection/quantification for this method were determined to be 50 and 100 ng/mL, respectively. The method was found to be linear from 500 ng/mL to 10,000 ng/mL (r(2)>0.995). The recovery of GHB was found to be greater than 75%. In this report, results of authentic urine samples analyzed for GHB by this method are presented. GHB concentrations in these samples were found to be range from less than 500 ng/mL to 5110 ng/mL.     

Liquid chromatography-tandem mass spectrometry analysis of anisodamine and its phase I and II metabolites in rat urine

A sensitive and specific method for the analysis of anisodamine and its metabolites in rat urine by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS) was developed. Various extraction techniques (free fraction, acid hydrolyses and enzyme hydrolyses) and their comparison were carried out for investigation of the metabolism of anisodamine. After extraction procedure the pretreated samples were injected on a reversed-phase C18 column with mobile phase (0.2 ml/min) of methanol/0.01% triethylamine solution (adjusted to pH 3.5 with formic acid) (60:40, v/v) and detected by MS/MS. Identification and structural elucidation of the metabolites were performed by comparing their changes in molecular masses (DeltaM), retention-times and full scan MS(n) spectra with those of the parent drug. At least 11 metabolites (N-demethyl-6beta-hydroxytropine, 6beta-hydroxytropine, tropic acid, N-demethylanisodamine, hydroxyanisodamine, anisodamine N-oxide, hydroxyanisodamine N-oxide, glucuronide conjugated N-demethylanisodamine, sulfate conjugated and glucuronide conjugated anisodamine, sulfate conjugated hydroxyanisodamine) and the parent drug were found in rat urine after the administration of a single oral dose 25mg/kg of anisodamine. Hydroxyanisodamine, anisodamine N-oxide and the parent drug were detected in rat urine for up 95 h after ingestion of anisodamine.     

Simultaneous determination of phenylbutazone and its metabolites in plasma and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the simultaneous determination of phenylbutazone and its metabolites, oxyphenbutazone and γ-hydroxyphenylbutazone, in plasma and urine. Samples were acidified with hydrochloric acid and extracted with benzene—cyclohexane (1:1, v/v). The extract was redissolved in methanol and chromatographed on a μBondapak C₁₅ column using a mobile phase of methanol—0.01 M sodium acetate buffer (pH 4.0) in a linear gradient (50 to 100% methanol at 5%/min; flow-rate 2.0 ml/min) in a high-performance liquid chromatograph equipped with an ultra-violet absorbance detector (254 nm). The detection limit for phenylbutazone, oxyphenbutazone and for γ-hydroxyphenylbutazone was 0.05 μg/ml.A precise and sensitive assay for the determination of phenylbutazone and its metabolites was established.     

Determination of myo-inositol in biological samples by liquid chromatography-mass spectrometry

A high-performance liquid chromatographic method using liquid-liquid extraction was developed for the determination of 1-(3-fluoro-4-hydroxy-5-mercaptomethyl-tetrahydrofuran-2-yl)-5-methyl-1H-pyrimidine-2,4-dione (l-FMAUS; I) in rat plasma and urine. A 100 microl aliquot of distilled water containing l-cysteine (100 mg/ml) was added to a 100 microl aliquot of biological sample. l-Cysteine was employed to protect binding between the 5'-thiol of I and protein in the biological sample. After vortex-mixing for 30s and adding a 50 microl aliquot of the mobile phase containing the internal standard (10 microg/ml of 3-aminophenyl sulfone), 1 ml of ethyl acetate was used for extraction. After vortex-mixing, centrifugation, and evaporating the ethyl acetate, the residue was reconstituted with a 100 microl aliquot of the mobile phase. A 50 microl aliquot was injected onto a C(18) reversed-phase column. The mobile phases, 50 mM KH(2)PO(4) (pH = 2.5):acetonitrile (85:15, v/v) for rat plasma and 50 mM KH(2)PO(4) (pH 2.5):acetonitrile:methanol (85:10:5, v/v/v) for urine samples, were run at a flow-rate of 1.2 ml/min. The column effluent was monitored by an ultraviolet detector set at 265 nm. The retention times for I and the internal standard were approximately 9.7 and 12.5 min, respectively, in plasma samples and the corresponding values in urine samples were 16.8 and 14.9 min. The quantitation limits of I in rat plasma and urine were 0.1 and 0.5 microg/ml, respectively.     

Rapid high-pressure liquid chromatographic determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in biological fluids

A rapid and sensitive high-pressure liquid chromatographic method for determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N¹⁰-methylpteroic acid has been developed. The assay is based on isocratic reversed-phase chromatography with siliceous microparticulate Spherisorb (5 μm, ODS, 15 × 0.4 cm i.d.) as stationary phase and a ternary solvent mixture of citrate-phosphate (0.05 m, pH 3.2)/methanol/tetrahydrofurane (80:16:4, v/v) as eluant. A precolumn of Perisorb (RP2, 30–40 μm, 3 × 0.4 cm i.d.) reasonably protects the analytical column against deterioration by the components of plasma or other biological fluids. Since the samples of plasma, urine, or cerebrospinal fluid are directly injected into the chromatographic system, the method is very rapid. Within 8 min as little as 50 ng of methotrexate and its metabolites per milliliter (10^?7m) can be measured with a precision better than 7%. Structural analogs of methotrexate do not interfere with the determination. There is a good correlation with the results of other methods, e.g., enzyme immunoassay or radioimmunoassay. The applicability for clinical monitoring in patient's plasma and urine is demonstrated.     

High-performance liquid chromatographic assay for melphalan in human plasma Application to pharmacokinetic studies

This paper describes a simple, rapid and reproducible high-performance liquid chromatographic method (HPLC) with ultraviolet absorbance detection for the analysis of melphalan in plasma. The HPLC column was an Ultrasphere ODS (5 μm) and the eluent was composed of methanol, purified water and acetic acid (49.5:49.5:1, v/v). The detection was performed at 261 nm. The method involved a simple treatment of the samples with methanol. The propylparaben was used as internnal standard. Linear detection response was obtained for concentrations ranging from 50 to 2500 ng/ml. Recovery from plasma proved to be more than 90%. Precision, expressed as C.V., was in the 0.5 to 9% range. Accuracy ranged from 95 to 102%. This method was used to determine the pharmacokinetic parameters of melphalan following high-dose (140 mg/m²) intravenous administration in patients with advanced malignancies undergoing peripheral blood hematopoietic progenitor-cell transplantation.     

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition

A high-performance liquid chromatographic method for the measurement of bumetamide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C₁₈ column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C₁₈ Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol—water—glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5–2000 ng/ml.     

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS—methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS—5% methanol (v/v) at 50°C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.     

Control of propranolol intake by direct chromatographic detection of alpha-naphthoxylactic acid in urine

A rapid chromatographic procedure with a C18 column, a mobile phase of 0.15 M sodium dodecyl sulfate (SDS)-10% (v/v) 1-propanol at pH 3 (0.01 M phosphate buffer), and fluorimetric detection, is reported for the control of propranolol (PPL) intake in urine samples, which are injected directly without any other treatment than filtration. The peak of PPL was only observed in samples taken a few hours after ingestion of the drug due to its extensive conjugation and metabolisation. The detection of several unconjugated PPL metabolites was therefore considered: desisopropylpropranolol (DIP), propranolol glycol (PPG), alpha-naphthoxylactic acid (NLT) and alpha-naphthoxyacetic acid (NAC). NLT showed the best characteristics: it eluted at a much shorter retention time than PPL, its concentration in urine samples was greater and it did not present any interference from endogeneous compounds in urine, common drugs or drugs administered in combination with PPL. The limit of quantification, measured as the concentration of analyte providing a relative standard deviation of 20%, was 24 ng/ml, and the day-to-day imprecision was below 4% for concentrations above 200 ng/ml. The procedure allows the routine control of PPL at therapeutic urine levels. Urinary excretion studies showed that the detection of NLT is possible at least up to 20-30 h after oral administration.     

Simultaneous determination of norepinephrine,serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.     

Direct and simultaneous analysis of loxoprofen and its diastereometric alcohol metabolites in human serum by on-line column switching liquid chromatography and its application to a pharmacokinetic study

A simple, rapid, and accurate column-switching liquid chromatography method was developed and validated for direct and simultaneous analysis of loxoprofen and its metabolites (trans- and cis-alcohol metabolites) in human serum. After direct serum injection into the system, deproteinization and trace enrichment occurred on a Shim-pack MAYI-ODS pretreatment column (10 mm x 4.6 mm i.d.) by an eluent consisting of 20 mM phosphate buffer (pH 6.9)/acetonitrile (95/5, v/v) and 0.1% formic acid. The drug trapped by the pretreatment column was introduced to the Shim-pack VP-ODS analytical column (150 mm x 4.6 mm i.d.) using acetonitrile/water (45/55, v/v) containing 0.1% formic acid when the 6-port valve status was switched. Ketoprofen was used as the internal standard. The analysis was monitored on a UV detector at 225 nm. The chromatograms showed good resolution, sensitivity, and no interference by human serum. Coefficients of variations (CV%) and recoveries for loxoprofen and its metabolites were below 15 and over 95%, respectively, in the concentration range of 0.1-20 microg/ml. With UV detection, the limit of quantitation was 0.1 microg/ml, and good linearity (r = 0.999) was observed for all the compounds with 50 microl serum samples. The mean absolute recoveries of loxoprofen, trans- and cis-alcohol for human serum were 89.6 +/- 3.9, 93.5 +/- 3.2, and 93.7 +/- 4.3%, respectively. Stability studies showed that loxoprofen and its metabolites in human serum were stable during storage and the assay procedure. This analytical method showed excellent sensitivity with small sample volume (50 microl), good precision, accuracy, and speed (total analytical time 18 min), without any loss in chromatographic efficiency. This method was successfully applied to the pharmacokinetic study of loxoprofen in human volunteers following a single oral administration of loxoprofen sodium (60 mg, anhydrate) tablet.     

Enantioselective analysis of (R)- and (S)-atenolol in urine samples by a high-performance liquid chromatography column-switching setup

An HPLC column-switching method for the enantioselective determination of (R,S)-atenolol in human urine was developed and validated. Diluted urine samples were injected onto a LiChrospher ADS restricted access column and atenolol was separated from most of the matrix components using 0.01 M Tris buffer. The atenolol peak was sharpened by a step gradient of 30% acetonitrile and the atenolol-containing fraction was switched onto an enantioselective column. Separation of the atenolol enantiomers was carried out on a Chirobiotic T (Teicoplanin) column using acetonitrile–methanol–acetic acid–triethylamine (55:45:0.3:0.2, v/v/v/v) as eluent. Detection of the effluent was performed by fluorescence measurement. Several experiments were carried out to suppress the high blank reading, which was efficiently achieved using Tris buffer in the first dimension. For the enantioselective analysis of (R)- and (S)-atenolol in plasma under the same conditions the sample capacity of the ADS column is considerably lower.     

Analysis of barbiturates in blood by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay for the identification and quantification of barbiturates in blood at therapeutic levels has been developed. An ODS-silica column is used with an eluent of 40% methanol at pH 8.5. The barbiturates are detected at 240 nm. The sample preparation procedure involves extraction of unfractionated blood (100 μl) with hexane—diethyl ether (50:50, v/v) and is very rapid. Talbutal is used as an internal standard.The method has been applied to the determination of five barbiturates (amylobarbitone, butobarbitone, cyclobarbitone, pentobarbitone and quinalbarbitone) in blood after therapeutic doses of the drugs. An application of the HPLC assay to forensic casework is demonstrated.     

Determination of methylguanidine in plasma and urine by high-performance liquid chromatography with fluorescence detection following postcolumn derivatization

A high-performance liquid chromatographic method was developed for the determination of methylguanidine in biological fluids. Methylguanidine and the internal standard were isolated from plasma by cation-exchange solid-phase extraction prior to chromatographic analysis. Urine samples were diluted and injected directly onto the analytical column. Chromatographic separation was carried out on an Ultrasil cation-exchange column using a mixture of methanol and monochloroacetate (15/85, v/v) as the mobile phase. Postcolumn derivatization of methylguanidine was carried out using alkaline ninhydrin reagent and the resulting fluorescent product was detected on-line. The method was specific, sensitive, reproducible, and linear over a wide a range of concentrations. The lower limit of detection for methylguanidine in plasma and urine was 1 and 100 ng/ml, respectively. The method was successfully employed for quantification of the levels of methylguanidine in normal and uremic human subjects, normal dogs, and dogs with ischemic-induced acute or spontaneous chronic renal failure.     

Determination of 1-hydroxypyrene in children urine using column-switching liquid chromatography and fluorescence detection

This study developed an acid hydrolysis method instead of using enzyme extraction, equipped with column-switching system for the pretreatment of samples, in the determination of 1-hydroxypyrene in the urine from children and pyrene in airborne particulates. We collected both types of samples from areas near a petrochemical industry and rural areas as reference. Samples were first treated with acid hydrolysis and followed by solvent extraction prior to being injected into the separation system for the determination with high performance liquid chromatography and fluorescence. A column-switching system was on-line with a C18 separation column to remove matrix interference and obtain a stable baseline of the chromatogram. The eluent used to separate the 1-hydroxypyrene was 60% (v/v) aqueous acetonitrile solution. A fluorescence detector was used to monitor 1-hydroxypyrene at lambdaex = 348 nm and lambdaem = 388 nm, and pyrene at lambdaex = 331 nm and lambdaem = 390 nm. Both calibration graphs were linear with very good correlation coefficients (r > 0.999) and the detection limits were ca. 2pg (5ng/l). Results showed that there was a significant association between 1-hydroxypyrene levels in urine specimens and pyrene levels in airborne particulate samples (r = 0.68, P < 0.05). The average levels of pyrene in the particulates (0.18 versus 0.09ng/m3) and of 1-hydroxypyrene in urine specimens (155.9 versus 110.2ng/g creatinine) were higher for the petrochemical area than for the rural area. This method is stable and sensitive for measuring polycyclic aromatic hydrocarbons in environmental samples.     

Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat.

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and alpha-phenylethylamine as an internal standard (IS). A sample (50 microL) of rat urine was added to 5 microL IS and 100 microL 100 mmol/L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 microL 75 mmol/L borate buffer (pH 8.5) and 50 microL 20 mmol/L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80 degrees C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN/CH3OH/10 mmol/L imidazole-HNO3 buffer (pH 7.0) (= 45:5:50, v/v/v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5-15 ng/mL detection limits at a S/N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg/kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13-160.1 and 0.17-10.9 microg/mL, respectively.