Determination of ondansetron and its hydroxy metabolites in human serum using solid-phase extraction and liquid chromatography/positive ion electrospray tandem mass spectrometry

Ondansetron and its hydroxylated metabolites were determined in human serum using solid-phase extraction (SPE) and liquid chromatography/positive ion electrospray tandem mass spectrometry. Pyrimethamine was used as the internal standard. The analytes were eluted from the SPE cartridge using 2 x 1 ml of methanol containing 0.5% triethylamine, evaporated under vacuum and the residue was reconstituted in the mobile phase. The liquid chromatographic separation was achieved on a silica column using a mobile phase of aqueous 20 mM ammonium acetate (pH 4.7)-acetonitrile (85 : 15, v/v) at a flow-rate of 0.4 ml min(-1). The method was linear over the range 1-500 ng ml(-1) for ondansetron and each of the metabolites in human serum. The intra-day accuracy was better than 9.1% and the precision was <10.3%; the inter-day accuracy was better than 9.5% and the precision was <12.6%. The limit of detection was 250 pg ml(-1) based on a signal-to-noise ratio of 3. The absolute recovery from serum for all analytes was >90%.     

Liquid chromatographic analysis of clonazepam in human serum with solid-phase (Bond-Elut) extraction

A simple, sensitive, selective and precise liquid-column chromatographic assay for clonazepam is described, in which 1 ml of serum containing 50 micrograms/l methylclonazepam as an internal standard is extracted by elution from a Bond-Elut column with 400 microliter of methanol. An aliquot of the eluate is injected on to a reversed-phase column and eluted with a mobile phase of acetonitrile--phosphate buffer (30:70) at a flow-rate of 2 ml/min at a column temperature of 50 degrees C. Detection is at 254 nm. Chromatography is complete in 12 min. A sensitivity of 2 ng/ml is attained when 1 ml of serum is extracted. Analytical recovery of the clonazepam added to serum ranged from 91% to 99% with a coefficient of variation of 6.0%. This assay for clonazepam has good precision, with coefficients of variation of 11% at 15 ng/ml and 2.6% at 50 ng/ml. There was no interference from any of the commonly used antiepileptics.     

Determination of malotilate and its metabolites in plasma and urine

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.     

Simultaneous determination of haloperidol and its metabolite, reduced haloperidol, in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography.

A high-performance liquid chromatographic method was developed for the simultaneous determination of haloperidol and reduced haloperidol in human plasma, urine and rat tissue homogenates using bromperidol as an internal standard. The method involved extraction followed by injection of 50-80 microliters of the aqueous layer onto a C18 reversed-phase column. The mobile phase was 0.5 M phosphate buffer-acetonitrile-methanol (58:31:11, v/v/v) and the flow-rate was 0.6 ml/min. The column effluent was monitored by ultraviolet detection at 214 nm. The retention times for reduced haloperidol, haloperidol and bromperidol were 5.4, 7.2 and 8.4 min, respectively. The detection limits for haloperidol and reduced haloperidol in human plasma were both 0.5 ng/ml, and the corresponding values in human urine were both 5 ng/ml. The coefficients of variation of the assay were generally low (below 10.7%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or any drug tested were found.     

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 bumetanide 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.     

Liquid chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection

A liquid chromatographic method using a solid-phase extraction procedure for the quantification of sotalol in plasma and urine is described. Sotalol is eluted from an extraction column with ethyl acetate-acetonitrile (1:2) and, after separation by reversed-phase high-performance liquid chromatography on a mu Bondapak C18 column, is quantified by fluorescence detection at excitation and emission wavelengths of 240 and 310 nm, respectively. The method has been demonstrated to be linear over the concentration ranges 10-6000 ng/ml in plasma and 0.5-100 micrograms/ml in urine. Mean inter-assay accuracy of the method for plasma ranged from 93 to 100% and for urine from 102 to 114%; precision ranged from 0.5 to 1.6% for plasma over a concentration range of 200-4000 ng/ml and for urine from 0.7 to 2.0% at concentrations of 2-50 micrograms/ml. Mass spectrometry confirmed the presence of sotalol in isolated chromatographic fractions of plasma and urine extracts from subjects given sotalol orally.     

Determination of cefodizime in biological materials by high-performance liquid chromatography

Cefodizime (THR-221) is a new semi-synthetic cephalosporin. A high-performance liquid chromatographic method has been developed for the determination of cefodizime in biological materials. A plasma or serum sample was deproteinized with methanol and the resulting methanol eluate was concentrated to a volume of 0.5 ml. Urine and bile samples were diluted with buffer and each diluted sample was filtered. Faeces samples were homogenized and the supernate obtained after centrifugation was filtered. Visceral tissue samples were homogenized, the centrifuged supernate was deproteinized with methanol, and the methanol eluate was concentrated to a volume of 0.5 ml. Aliquots of each preparation were chromatographed on a reversed-phase column with an ion-pair chromatographic technique on a high-performance liquid chromatograph equipped with an UV detector set at 264 nm. The detection limits for cefodizime were 0.1 microgram/ml in plasma or serum, 0.3 microgram/ml in bile, and 0.5 microgram/ml in urine, 0.5 microgram/g in faeces and visceral tissue. This precise and sensitive assay for the determination of cefodizime is described, and its stability in several media is reported.     

Determination of aniracetam and its main metabolite, N-anisoyl-GABA, in human plasma by high-performance liquid chromatography

Two different reversed-phase high-performance liquid chromatographic methods for the determination of aniracetam (I) and its metabolite N-anisoyl-GABA (II) in human plasma are described. The procedure for I involves direct injection of plasma samples spiked with the internal standard on a clean-up column followed by reversed-phase chromatography on a C18 column. The limit of quantification was 5 ng/ml, using a 200-microliters specimen of plasma. The mean inter-assay precision of the method up to 800 ng/ml was 3%. The procedure for II involved liquid-liquid extraction of II and the internal standard from plasma with ethyl acetate, and reversed-phase chromatography on a C18 column. The limit of quantification was 50 ng/ml using a 0.5-ml plasma specimen. The mean inter-assay precision up to 50 micrograms/ml was 6%. The applicability and accuracy of the methods were demonstrated by the analysis of over 1000 plasma samples from two bioavailability studies in healthy volunteers.     

Determination of pemoline in plasma, urine and tissues by reversed-phase high-performance liquid chromatography

A simple and selective high-performance liquid chromatographic method with ultraviolet detection at 215 nm for the determination for pemoline in rat plasma, urine and tissues is described. Pemoline in the samples was extracted with methylene chloride at pH 10 and the organic phase was evaporated after adding 5-methyl-5-phenylhydantoin used as an internal standard. Pemoline and the internal standard were separated on a Kaseisorb LC C8-60-5 reversed-phase column. The limits of determination of pemoline in 0.1-0.2 ml of plasma, urine and tissue homogenates were 2, 100 and 20 ng, respectively. The method should be useful for studies of the pharmacokinetics and distribution of pemoline in small animals.     

Simultaneous determination of methylprednisolone and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt in human urine by high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic assay with ultraviolet detection at 243 nm has been developed for the quantitative determination of methylprednisolone (MP) and methylprednisolone 21-[8-[methyl-(2-sulfoethyl)amino]-8-oxooctanoate] sodium salt (MPSO) in human urine following therapeutic doses in humans. The assay procedure involves stabilization of urine samples by addition of disodium ethylenediaminetetraacetic acid (Na2EDTA) and ion-pair extractions of MPSO using tetraethylammonium chloride (TEACl) as the counter ion. After extracting both drugs and internal standard into chloroform, the extract was evaporated to dryness under nitrogen. The resulting residue was reconstituted in 200-500 microliters of mobile phase and chromatographed on an IBM C18 reversed-phase column (5 microns). The mobile phase was a mixture of water-acetonitrile-isopropanol (71.2:18.8:10.0, v/v) containing 75 microliters of 0.1 M hydrochloric acid and 0.450 g of TEACl per liter. Propyl p-hydroxybenzoate was used as an internal standard. The extraction efficiencies of MP and MPSO were greater than 90% using the ion-pairing agent TEACl. The chromatographic responses were linear up to about 200 micrograms/ml for MP and 80 micrograms/ml for MPSO and had sufficient precision and accuracy to provide quantitative data from human urine. The assay detection limit was about 8 ng/ml for MP and 25 ng/ml for MPSO in human urine. Stability studies in urine indicated that without Na2EDTA stabilization and at room temperature, rapid degradation of MPSO occurred in urine. Addition of EDTA to the urine specimen and storage at -70 degrees C increased the stability of MPSO, and little or no degradation was observed in urine stored for more than 60 days. The method has been used in the simultaneous determination of MP and MPSO in urine specimens obtained from a single-dose tolerance study of MPSO in normal male volunteers.     

Analytical and semi-preparative high-performance liquid chromatographic separation and assay of hydroxychloroquine enantiomers.

(+/-)-Hydroxychloroquine (HCQ) is an antimalarial and anti-arthritic drug which is administered as the racemate. An accurate, precise and sensitive high-performance liquid chromatographic assay was developed for the determination of HCQ enantiomers in samples from human plasma, serum, whole blood, and urine. After addition of (+/-)-chloroquine (internal standard), samples of blood component (0.5 ml) or urine (0.1 ml) were alkalinized and extracted with 5 ml of diethyl ether. After solvent evaporation the residues were derivatized with (+)-di-O-acetyl-L-tartaric anhydride at 45 degrees C for 30 min. The resulting diastereomers were then resolved using a C8 analytical column with a mobile phase consisting of 0.05 M KH2PO4 (pH 3)-methanol-ethanol-triethylamine (78:22:1:0.08). The ultraviolet detection wavelength was set at 343 nm. The derivatized HCQ enantiomers eluted in less than 40 min, free of interfering peaks. Excellent linear relationships (r2 > 0.997) were obtained between the area ratios and the corresponding plasma concentrations over a range of 12.5-500 ng/ml. The diastereomers could be hydrolysed using microwave energy and neutral pH, which enabled us to resolve the enantiomers on a semi-preparative (C18 column) scale. The method was suitable for the analysis and semi-preparative separation of HCQ enantiomers.     

High-performance liquid chromatographic determination of (S)-(-)-ofloxacin and its metabolites in serum and urine using a solid-phase clean-up

A sensitive and selective method for the simultaneous determination of (S)-(-)-ofloxacin [(S)-(-)-OFLX] and its metabolites in serum and urine was developed using isocratic high-performance liquid chromatography with a specific solid-phase extraction procedure. (S)-(-)-OFLX and its metabolites, desmethyl-(S)-(-)-OFLX and (S)-(-)-OFLX N-oxide, were eluted from a C8 solid-phase column with recoveries of more than 98%. These compounds were separated and determined by means of a reversed-phase column with fluorimetric detection. Validation studies showed that the results were linear for (S)-(-)-OFLX in serum over the range 10-1200 ng/ml and in urine over the range 1-200 micrograms/ml. Analysis for (S)-(-)-OFLX and its metabolites showed good precision and accuracy with a relative standard deviation of less than 6%.     

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 microliters 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 micrograms/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.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection.

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C18 column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 microliters of the mobile phase. A 10-microliters aliquot of the resulting solution is injected onto a 150 mm x 4.6 mm I.D. column packed with 5-microns octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E1 = +0.25 V and E2 = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90-95% which allows the determination of 0.5 microgram/l analyte.     

Determination of ramoplanin in human urine by high-performance liquid chromatography with automated column switching.

Ramoplanin is a novel glycolipodepsipeptide antibiotic, currently undergoing clinical trials. This method describes the determination of ramoplanin by direct injection of human urine into a coupled-column liquid chromatographic system. An internal-surface reversed-phase column has been used for on-line sample clean-up and enrichment. Analytical separation of ramoplanin and MDL 62,456 used as internal standard, has been achieved on a ABZ+ reversed-phase column with ammonium acetate buffer-acetonitrile-methanol according to a gradient profile. Analytes were detected by their UV absorbance at 270 nm. The limit of quantitation was 0.1 microgram/ml urine and the limit of detection was found to be 0.035 microgram/ml, corresponding to 13.7 pmol/ml. Linearity was determined in the range 0.1-2 micrograms/ml. Precision (relative standard deviation) ranged from 0.71 to 8.75% and the accuracy from -9.9 to 11.6%. Different human sources were tested and no interference between analytes and urine constituents was observed. The method is simple and rapid, requiring a total analysis time of 35 min per sample and reaching greater selectivity and accuracy than microbiological assays.     

Solid-phase extraction with liquid chromatography and ultraviolet detection for the assay of antidepressant drugs in human plasma

A solid-phase extraction (SPE) method for sample clean-up followed by a reversed-phase high-performance liquid chromatography (HPLC) procedure for the assay of five antidepressant drugs (trazodone, doxepin, desipramine, maprotiline and imipramine) is reported. The drugs were recovered from plasma buffered at a suitable pH using C18 Bond-Elut cartridges and mixtures of methanol-aqueous buffer as washing and elution solvents. The recoveries of the drugs using other sorbent materials (C8, C2, cyclohexyl, cyanopropyl and phenyl Bond Elut and copolymer HLB waters cartridges) were also examined. The selectivity of SPE was examined by using spiked plasma samples and the CH cartridge gave rise to the cleanest extracts. Cyclohexyl cartridges were conditioned successively with 2 ml of methanol and 1 ml of acetic acid-sodium acetate buffer (0.1 M, pH 4.0). Plasma sample was buffered at pH 4.0 and then applied to the sorbent. The washing step was performed subsequently with 1.5 ml of acetate buffer (0.1 M, pH 4.0), 100 microl of acetonitrile and 1 ml of methanol-acetate buffer (30:70, v/v). Finally, the analytes were eluted with 0.5 ml of methanol-acetate buffer (70:30, v/v). The extract was evaporated to dryness, reconstituted in mobile phase, and chromatographed on a reversed-phase C18 column with ultraviolet detection at 215 nm. The recoveries of trazodone, doxepin, desipramine, maprotiline and imipramine from spiked plasma samples using the CH cartridge were 58 2, 84 3, 83 3, 83 3 and 82 2%, respectively. The within-day and between-day repeatabilities were lower than 6% and 9%, respectively. The linearity of calibrations for the five antidepressants was between 0.005 and 2 microg/ml. The limits of detection were 1 ng/ml for trazodone, doxepin and desipramine and 2 ng/ml for maprotiline and imipramine.     

High-performance liquid chromatographic assay of pirlimycin in human serum and urine using 9-fluorenylmethylchloroformate

A reversed-phase high-performance liquid chromatographic (HPLC) assay method has been developed for determining pirlimycin in human serum and urine. The method involves chloroform extraction of pirlimycin free base followed by derivatization with 9-fluorenylmethylchloroformate to form a carbamate ester. The reaction is rapid, reproducible, and quantitative. 9-Fluorenylmethylchloroformate reacts with amines to form derivatives sensitive to both ultraviolet and fluorescence detection. Human serum and urine samples following 50-mg and 500-mg single oral doses of pirlimycin were analyzed. The samples were chromatographed on an RP-18 Spherisorb 5-micron, 250 X 4.6 mm I.D. reversed-phase HPLC column. The eluent for the serum assay was acetonitrile-water (58:42) containing 0.02% acetic acid, and for the urine assay was acetonitrile-methanol-tetrahydrofuran-water (48:2:1:49). Fluoranthene was used as an internal standard. The assay sensitivity by ultraviolet detection (lambda max = 264) was about 5 ng/ml and by fluorescence detection (lambda excitation = 270 nm, lambda emission = 300 nm) was 0.1 ng/ml. Statistical analysis indicates an average drug recovery of 101 +/- 4.2% from serum and 102.0 +/- 2.62% from urine.     

Liquid chromatographic determination of pinacidil, a new antihypertensive drug, and its major metabolite, pinacidil N-oxide, in plasma

Two procedures are described, one for the determination of pinacidil, the other for the determination of both pinacidil and its metabolite, pinacidil N-oxide, in plasma. When only parent drug levels are required, the plasma proteins are precipitated with acetonitrile, the solids discarded and the supernatant is evaporated to dryness. The residue is then reconstituted for analysis. For the determination of both drug and metabolite, the analytes are selectively retained from plasma on a solid-phase extraction column and eluted with methanol. After evaporation to dryness, the residue is reconstituted in mobile phase. Both procedures utilize reversed-phase liquid chromatographic separations with ultraviolet detection. The limits of detection are 10 ng/ml pinacidil in plasma and 5 ng/ml each of pinacidil and pinacidil N-oxide in plasma for the two procedures, respectively.     

Analysis of lorazepam in rat brain using liquid/liquid and solid-phase extraction in combination with high performance liquid chromatography

A method for the determination of lorazepam in rat brain is described using liquid/liquid and solid-phase extraction, followed by high performance liquid chromatography. After addition of chlordiazepoxide as the internal standard, 100 mg brain tissue was homogenized and incubated with alkaline protease. Lorazepam and chlordiazepoxide were extracted three times with toluene. After treatment through a C18-Bond Elut column, lorazepam and chlordiazepoxide were analyzed isocratically on a reversed-phase column with a mobile phase consisting of methanol +0.025 M sodium phosphate buffer (66:34, v/v). The eluted drugs were monitored by their absorption at 240 nm. The sensitivity limit of this method was 10 ng of lorazepam per 100 mg of brain tissue sample. The standard curve was linear over the range of 20 to 200 ng lorazepam. The coefficient of variation for day-to-day precision established by 21 replicate analyses was 4.5 to 13.6%.     

Determination of cetirizine in human urine by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the determination of the histamine H1-receptor antagonist cetirizine in human urine was developed. Cetirizine and the internal standard are extracted from acidified (pH 5) urine (0.5 ml) into chloroform and the organic layer is evaporated to dryness. The residue is chromatographed on a Spherisorb 5ODS-2 column using Pic A (5 mM aqueous tetrabutylammonium phosphate)-methanol-tetrahydrofuran (33:65:2, v/v) as the mobile phase with ultraviolet detection (230 nm). The calibration graph is linear from 0.1 to 10 micrograms/ml and using 0.5 ml of urine the detection limit is 20 ng/ml. The within-run relative standard deviation is less than 6% and the accuracy is within 10% of the theoretical value at concentrations between 0.1 and 10 micrograms/ml in urine. There is a good correlation (r = 0.99606) with a previously described capillary gas chromatographic assay.