Simultaneous determination of codeine, norcodeine and morphine in biological fluids by high-performance liquid chromatography with fluorescence detection

A novel high-performance liquid chromatographic method for the determination of codeine, norcodeine and morphine in plasma and urine has been developed. The compounds were separated on a cyano column (15 cm x 4.6 mm, 5 microns particle size) using a mobile phase of acetonitrile-triethylamine-distilled water (4:0.1:95.9, v/v) pH 3.1 and then determined by fluorescence detection. Calibration curves in the range 5-200 ng/ml for plasma and 0.1-10 micrograms/ml for urine were linear and passed through the origin. The imprecision and inaccuracy of the assay were less than 10% and the limits of detection were 2 ng/ml for all three compounds in human plasma.     

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%.     

Analytical procedure for the determination of rufloxacin, a new pyridobenzothiazine, in human serum and urine by high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantification of rufloxacin in human serum and urine has been developed and validated. The compounds, rufloxacin and internal standard, are extracted from buffered serum and urine using dichloromethane. They are then separated on an anion-exchange column using 0.05 M phosphate buffer-acetonitrile (80:20, v/v). The eluate is quantified by measuring the ultraviolet absorbance at 296 nm. The lower limit of detection for the analyte is 0.1 microgram/ml in serum and 0.05 micrograms/ml in urine. The method is linear from 0.3 to 10 micrograms/ml for serum and 0.1 to 10 micrograms/ml for urine. The method has been applied in a pharmacokinetic study in volunteers.     

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.     

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.     

Determination of pioglitazone in dog serum using solid-phase extraction and high-performance liquid chromatography with ultraviolet (229 nm) detection

An analytical method is described for the determination of the free base of pioglitazone hydrochloride (U72, 107A, AD-4833) in dog serum. The method used solid-phase extraction of pioglitazone from serum followed by high-performance liquid chromatographic analysis on an octadecylsilane column with an eluent of acetonitrile-water (41:59, v/v) containing 1.2 ml/l acetic acid (pH 6.0 +/- 0.05). The column effluent was monitored at 229 nm. The analytical procedure has a linear range of 25 ng/ml to 20 micrograms/ml, a minimum quantifiable level of 25 ng/ml, absolute recovery of greater than 90% (n = 15), and precision of less than or equal to 8.8% (n = 45). The method was used in a preliminary dose proportionality study in the dog.     

Determination of cetirizine in serum using reversed-phase high-performance liquid chromatography with ultraviolet spectrophotometric detection.

A method using reversed-phase high-performance liquid chromatography with ultraviolet detection for the determination of ceterizine in serum is described. The method is sensitive down to 50 ng/ml (250-microliter loop). Sample preparation involves only serum deproteination with perchloric acid and injection of the centrifuged supernatant. Elution is at pH 2.5 with acetonitrile-methanol-0.05 M phosphate buffer (33:9:58, v/v) on a 25 cm x 4.6 mm I.D. Spherisorb S5 ODS2 column. Detection is at 211 nm, its lambda max. For levels above 300 ng/ml the serum sample size is 100 microliter and a 200-microliter sample is necessary for concentrations less than 300 ng/ml. At the 2 micrograms/ml concentration the intra-assay relative standard deviation is better than 2.2%, whilst the inter-assay deviation is 2.6% over eight samples. At 200 ng/ml the intra-assay relative standard deviation is 6% over seven samples. Detector response is linear from 100 ng/ml to 10 micrograms/ml (100-microliter loop).     

Determination of temazepam and temazepam glucuronide by reversed-phase high-performance liquid chromatography.

A rapid and sensitive method for extracting temazepam from human serum and urine is presented. Free temazepam is extracted from plasma and urine samples using n-butyl chloride with nitrazepam as the internal standard. Temazepam glucuronide is analyzed as free temazepam after incubating extracts with beta-glucuronidase. Separation is achieved using a C8 reversed-phase column with a methanol-water-phosphate buffer mobile phase. An ultraviolet detector operated at 230 nm is used and a linear response is observed from 20 ng/ml to 10 micrograms/ml. The limit of detection is 15.5 ng/ml and the limit of quantitation is 46.5 ng/ml. Coefficients of variation are less than 10% for concentrations greater than 50 ng/ml. Application of the methodology is demonstrated in a pharmacokinetic study using eight healthy male subjects.     

Simultaneous Determination of Hydrochlorothiazide and Reserpine in Human Urine by LC with a Simple Pre-Treatment

A simple, selective and sensitive reversed-phase high performance liquid chromatography method for simultaneous analysis of hydrochlorothiazide and reserpine in human urine was developed and subjected to primary pharmacokinetic study. After a simple protein precipitation using methanol and extraction with ethyl acetate, the analytes were separated on an Elite C(18) column at a flow rate of 0.8 mL min(-1). The mobile phase was composed of acetonitrile (A) and 0.2% ammonium chloride solution (B) for a gradient elution starting at A:B at 30:70, v/v for 0~6 min, linearly raising the percent of A from 30% to 50% (6~9 min) and ending at 50:50, v/v (9~25 min). The standard curves were linear over the range of 0.05-20 μg mL(-1) for hydrochlorothiazide and 0.02-5.0 μg mL(-1) for reserpine, respectively (r > 0.999). The limit of detection (LOD) and the limit of quantification (LOQ) were 5.5 ng mL(-1) and 18.2 ng mL(-1) for hydrochlorothiazide, and 7.1 ng mL(-1) and 23.6 ng mL(-1) for reserpine, respectively. The recoveries for both analytes were above 89.0±1.35%. The intra-day and inter-day precision for hydrochlorothiazide were less than 1.91% and 1.38%, and those for reserpine were below 1.61% and 2.64%, respectively. The method indicated good performance in terms of specificity, linearity, detection and quantification limits, precision and accuracy, and it was employed successfully for the simultaneous determination of hydrochlorothiazide and reserpine in human urine samples.     

High-performance liquid chromatographic determination of cefonicid in human plasma and urine

A high-performance liquid chromatographic assay for determination of cefonicid concentrations in human plasma and urine samples has been developed using cefazolin as an internal standard. For the analysis of plasma samples two calibration curves were utilized covering the cefonicid concentration ranges of 0.05-1.0 microgram/ml and 1.0-50.0 micrograms/ml, respectively. Coefficients of variation of 7.4% or less were obtained for cefonicid concentrations of 0.05-50.0 micrograms/ml. Mean bias was +6.0% at 0.05 micrograms/ml cefonicid and between -2.1% and +1.6% for 1.0-50.0 micrograms/ml cefonicid. Plasma samples containing 30 ng/ml cefonicid could be well distinguished from blank plasma samples. Urine samples were analysed by using a calibration curve for cefonicid concentrations between 1.0 and 50.0 micrograms/ml. ranged from 8.6% at a cefonicid concentration of 1.0 microgram/ml to 0.5% at 50.0 micrograms/ml with a mean bias between -3.0% and +0.3%.     

Quantification of the plant-derived hallucinogen Salvinorin A in conventional and non-conventional biological fluids by gas chromatography/mass spectrometry after Salvia divinorum smoking

A gas chromatography method with mass spectrometric detection is described for the determination of Salvinorin A, the main active ingredient of the hallucinogenic mint Salvia divinorum. The method was validated in plasma, urine, saliva and sweat using 17-alpha-methyltestosterone as internal standard. The analytes were extracted from biological matrices with chloroform/isopropanol (9:1, v/v). Chromatography was performed on a 5% phenyl methyl silicone capillary column and analytes were determined in the selected ion monitoring mode. The method was validated over the concentration range 0.015-5 microg/mL plasma, urine and saliva and 0.01-5 microg/patch in the case of sweat. Mean recoveries ranged between 77.1 and 92.7% for Salvinorin A in different biological matrices, with precision and accuracy always better than 15%. The method was applied to the analysis of urine, saliva and sweat from two consumers after smoking 75 mg plant leaves to verify the presence of the active ingredient of S. divinorum in human biological fluids as a biomarker of plant consumption. Salvinorin A was detected in urine (2.4 and 10.9 ng/mL) and saliva (11.1 and 25.0 ng/mL), but not in sweat patches from consumers.     

High-performance liquid chromatographic determination of BRB-I-28, a novel antiarrhythmic agent, in dog plasma and urine.

A sensitive reversed-phase high-performance liquid chromatographic (HPLC) technique with ultraviolet detection has been developed to determine the concentration of BRB-I-28 (I), a novel antiarrhythmic agent, in dog plasma and urine. The mobile phase was acetonitrile-methanol-37.5 mM phosphate buffer, pH 6.8-triethylamine (50:50:75:0.1, v/v). The compound was extracted from dog plasma and urine with chloroform after alkalinization with sodium hydroxide. The extraction recovery was 83% from plasma and 84% from urine. Good linearity (r > 0.996) was observed throughout the ranges 0.1-12.0 micrograms/ml (plasma) and 0.1-8.0 micrograms/ml (urine). Intra- and inter-assay variabilities were less than 4%. The lower limit of quantitation was 0.08 microgram/ml in either plasma or urine. HPLC analysis of plasma and urine samples from a dog treated with I has demonstrated that the method was accurate and reproducible.     

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.     

Sensitive, rapid and easy analysis of three catecholamine metabolites in human urine and serum by liquid chromatography tandem mass spectrometry

A sensitive and easy analytical method for catecholamine metabolites including 4-hydroxy-3-methoxyphenylglycol sulfate (HMPG sulfate), vanillylmandelic acid (VMA) and homovanillic acid (HVA) determination was developed based on liquid chromatography-tandem mass spectrometry in a negative multiple reaction monitoring mode. The analytes were rapidly separated on a reversed-phase Waters Xbridge C18 column (150 × 2.1 mm i.d.) with the mobile phase of 15% (v/v) acetonitrile containing 2 mM ammonium formate and 85% (v/v) formic acid solution (0.05%, v/v). Mass spectrometric conditions, such as characteristic fragmentations and quantification ion transitions, both with chromatographic conditions including separation column type and mobile phase composition, were systematically investigated to get optimal sensitivity and specificity. The limits of detection were in the range of 0.03-0.7 ng/mL for the targets. Recovery rates of spiked urine samples with three different concentration levels (low, middle and high) were above 86% with precisions less than 5.7%. For serum analysis, acetonitrile chosen both as protein precipitation reagent and extraction solvent facilitates to reduce matrix effects. Recovery rates of spiked serum sample were in the range of 90.6% to 111.1% for three targets. The intra-day and inter-day precisions were satisfactory less than 8.7%. This proposed method was successfully applied to determine HMPG sulfate, HVA and VMA present in human urine and serum.     

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.     

High-performance liquid chromatographic analysis of amphotericin B in plasma, blood, urine and tissues for pharmacokinetic and tissue distribution studies.

A sensitive and reproducible high-performance liquid chromatographic method was developed to assay ampherotericin B in plasma, blood, urine and various tissue samples. Amphotericin B was isolated from each sample matrix by solid-phase extraction (Bond-Elut). The eluate from Bond-Elut containing amphotericin B was injected onto a reversed-phase C18 column (Waters, mu Bondpak, 10 microns, 300 mm x 3.9 mm I.D.) with a mobile phase of 45% acetonitrile in 2.5 mM Na2EDTA at 1 ml/min. Detection of amphotericin B was by ultraviolet absorption at 382 nm. Blood and tissues were homogenized and extracted with methanol prior to Bond-Elut extraction. The extraction efficiencies of amphotericin B from plasma, blood and tissues were approximately 90, 70 and 75%, respectively. The sensitivity of the assay was less than or equal to 5 ng/ml for plasma, less than or equal to 25 ng/ml for blood, 2.5 ng/ml for urine and 50 ng/g for tissues. The linearity of the assay method was up to 2.5 micrograms/ml for plasma, 5 micrograms/ml for blood, 500 ng/ml for urine and 500 micrograms/g for tissues. The assay was reproducible with an intra-day coefficient of variation (C.V., n = 3) of less than 5% in general for plasma, blood and tissues. The inter-day C.V. of the assay was less than 5% for plasma (n = 5), less than 10% for blood (n = 4) and less than 5% for tissues (n = 3). The overall variability in the urine assay was generally less than 10%. This method has demonstrated significant improvement in the sensitivity and reproducibility in assaying amphotericin B in plasma and especially in blood, urine and tissues. We have employed this assay to compare the pharmacokinetic and tissue distribution profiles of amphotericin B in rats and dogs following administration of Fungizone and ABCD (amphotericin B-cholesteryl sulfate colloidal dispersion), a lipid-based dosage form. In addition, the assay method for plasma and urine samples can also be applied to pharmacokinetics studies of amphotericin B in man.     

Direct determination of chlorpyrifos and its main metabolite 3,5, 6-trichloro-2-pyridinol in human serum and urine by coupled-column liquid chromatography/electrospray-tandem mass spectrometry

A rapid and sensitive automated coupled-column liquid chromatography/electrospray tandem mass spectrometry (LC/LC/ES-MS/MS) method has been developed for the quantitation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol (TCP) in both human serum and urine. Human serum was first protein precipitated with acetonitrile, while urine was directly injected into the coupled-column system. A 10 microL aliquot was then analyzed using as first separation column a Discovery C18 5 microm 50 x 2.1 mm; the fraction containing the analyte was transferred on-line to the second column consisting of a ABZ+ 5 microm 100 x 2.1 mm, which was connected to the electrospray source (Z-spray) of a Quattro LC triple-quadrupole instrument. Chlorpyrifos was detected in positive ion mode using four multi reaction monitoring (MRM) transitions while TCP was measured in negative ion mode using three pseudo-MRM transitions. The clean-up performed by the coupled-column approach avoids the use of an internal standard for the correct quantitation of both analytes, and the highly automated procedure renders a sample throughput of more than 100 samples per day. Both compounds can be determined using the same set-up, the only difference in the procedure being the composition of the first mobile phase. The method has proved to be fast, reliable and sensitive, yielding calibration curves for both analytes with correlation coefficients greater than 0.9995. The repeatability and reproducibility at 5 and 50 ng/mL was lower than 8%. The accuracy and precision were evaluated by means of recovery experiments from fortified serum (5-50 ng/mL) and urine (1-10 ng/mL) samples, obtaining satisfactory recoveries for both compounds (87-113% in serum, and 98-109% in urine), with coefficients of variation (CVs) less than 10%. The detection limits were similar for chlorpyrifos and metabolite: 1.5 ng/mL in serum, and 0.5 ng/mL in urine, where no sample handling took place. The validated procedures provide excellent tools for the specific assessment of occupational exposure to the organophosphorus pesticide chlorpyrifos, throughout the analysis of both human serum and urine, and it is more selective and sensitive than the current assay based on the measurement of the decrease in the cholinesterase activity.     

Analysis of lamotrigine and lamotrigine 2-N-glucuronide in guinea pig blood and urine by reserved-phase ion-pairing liquid chromatography.

Lamotrigine is an investigational anticonsulvant drug undergoing clinical trials. A simultaneous assay was developed to quantitate lamotrigine and its major metabolite, lamotrigine 2-N-glucuronide, from guinea pig whole blood. The extraction procedure and reversed-phase high-performance liquid chromatographic (HPLC) assay employed sodium dodecylsulfate (SDS) as an ion-pairing reagent to selectively separate lamotrigine and lamotrigine 2-N-glucuronide from endogenous blood components, other anti-convulsant drugs, and their metabolites. The mobile phase was composed of acetonitrile-50 mM phosphoric acid (pH 2.2) containing 10 mM SDS (33:67, v/v), and components were detected at 277 nm. The total coefficients of variance (C.V.) for the blood assay were less than or equal to 9.4% for lamotrigine (0.25-20.0 micrograms/ml) and less than or equal to 13.4% for the glucuronide metabolite (0.25-10.0 micrograms/ml). Separate assays for lamotrigine and its glucuronide in urine were developed. In order to quantitate low levels of lamotrigine in guinea pig urine, lamotrigine was extracted with tert.-butyl methyl ether-ethyl acetate (1:1). The total C.V. for lamotrigine quantitation in urine was less than or equal to 7.5% (0.10-10.0 micrograms/ml). For the determination of lamotrigine 2-N-glucuronide, urine was diluted with an SDS-phosphoric acid buffer (1:4) and injected directly onto the HPLC system, total C.V. less than or equal to 4.2% (0.5-50 micrograms/ml).     

Direct enantiomeric resolution of disopyramide and its metabolite using chiral high-performance liquid chromatography. Application to stereoselective metabolism and pharmacokinetics of racemic disopyramide in man

A method for the simultaneous determination of disopyramide and mono-N-desisopropyldisopyramide enantiomers extracted from human plasma and urine is presented. Separation and quantitation were carried out using two columns coupled in series, and UV detection at 254 nm. First, the racemates of the two compounds were separated using a reversed-phase column, and then the enantiomers were separated using a stereoselective column packed with human alpha 1-acid glycoprotein. The mobile phase was 8 mM phosphate buffer, pH 6.20-2-propanol (92:8, v/v). The coefficients of variation (%) for the plasma daily determination were 6.7% for R(-)- and S(+)-disopyramide at drug levels of 1.5 micrograms/ml, and 8.5% and 7.7% for R(-)- and S(+)-mono-N-desisopropyldisopyramide, respectively, at drug levels of 0.375 micrograms/ml. The method has allowed the study of stereoselective metabolism and pharmacokinetics of disopyramide after oral administration as a racemate.     

High-performance liquid chromatographic analysis of isoxicam in human plasma and urine

A sensitive, selective, and rapid high-performance liquid chromatographic procedure was developed for the determination of isoxicam in human plasma and urine. Acidified plasma or urine were extracted with toluene. Portions of the organic extract were evaporated to dryness, the residue dissolved in tetrahydrofuran (plasma) or acetonitrile (urine) and chromatographed on a mu Bondapak C18 column preceded by a 4-5 cm X 2 mm I.D. column packed with Corasil C18. Quantitation was obtained by UV spectrometry at 320 nm. Linearity in plasma ranged from 0.2 to 10 micrograms/ml. Recoveries from plasma samples seeded with 1.8, 4 and 8 micrograms/ml isoxicam were 1.86 +/- 0.077, 4.10 +/- 0.107 and 8.43 +/- 0.154 micrograms/ml with relative standard deviations of 3.3%, 2.5% and 5.4%, respectively. The linearity in urine ranged from 0.125 to 2 micrograms/ml. The precision of the method was 3.3-9.0% relative standard deviation over the linear range.