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.     

Ion-Pair Liquid Chromatographic Analysis of Phenylpropanolamine in Plasma and Urine by Post-Column Derivatization with O-Phthalaldehyde

Abstract

A high pressure Liquid chromatographic analysis of phenylpropano Lamine in plasma and urine by post-column derivatzaition with o-phthalaldehyde is described. Plasma samples are extracted with methylene chloride under alkaline conditions. Urine is diluted with mobile phase without extraction. Using fluorescence detection, the method is sufficiently sensitive (2 ng/ml in 0.5 ml of plasma and 0.5 mcg/ml in 0.2 ml of urine) so that phenylpropano lamine concentrations in plasma or urine may be measured for up to 24 hours following a 75 mg oral dose. Coefficients of variation for inter-day and intra-day precision are less than 10%.     

Determination of famotidine in low-volume human plasma by normal-phase liquid chromatography/tandem mass spectrometry

A rapid, sensitive and robust assay procedure using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for the determination of famotidine in human plasma and urine is described. Famotidine and the internal standard were isolated from plasma samples by cation-exchange solid-phase extraction with benzenesulfonic acid (SCX) cartridges. The urine assay used direct injection of a diluted urine sample. The chromatographic separation was accomplished by using a BDS Hypersil silica column with a mobile phase of acetonitrile-water containing trifluoroacetic acid. The MS/MS detection of the analytes was set in the positive ionization mode using electrospray ionization for sample introduction. The analyte and internal standard precursor-product ion combinations were monitored in the multiple-reaction monitoring mode. Assay calibration curves were linear in the concentration range 0.5--500 ng ml(-1) and 0.05--50 microg ml(-1) in plasma and urine, respectively. For the plasma assay, a 100 microl sample aliquot was subjected to extraction. To perform the urine assay, a 50 microl sample aliquot was used. The intra-day relative standard deviations at all concentration levels were <10%. The inter-day consistency was assessed by running quality control samples during each daily run. The limit of quantification was 0.5 ng ml(-1) in plasma and 0.05 microg ml(-1) in urine. The methods were utilized to support clinical pharmacokinetic studies in infants aged 0-12 months.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid-liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5-2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

High-performance liquid chromatographic assay for dilevalol in human plasma and urine using a PRP-1 column and fluorimetric detection

A single high-performance liquid chromatographic (HPLC) assay for the quantitative determination of dilevalol, the R,R isomer of labetalol, was developed for both plasma and urine. A significantly improved limit of detection for dilevalol in plasma was accomplished by extensive modification of an HPLC assay originally developed in our laboratory for labetalol. This simplified method is readily adaptable to urine and represents the first reported HPLC assay for the quantitative determination of dilevalol in this biofluid. Drug was recovered from plasma or urine by partition into diethyl ether under mildly alkaline conditions and back-extraction into dilute acid. Reversed-phase separation of dilevalol and the internal standard was accomplished on a 150 X 4.1 mm column commercially packed with a spherical (5 micron) macroporous copolymer (PRP-1). No interferences were observed in extracts obtained from drug-free plasma or urine. Selectivity for dilevalol in the presence of other beta-blockers was established. This method demonstrated a linear detector response to concentrations of unchanged drug typically observed in urine and plasma following once-a-day treatment with dilevalol hydrochloride (100-800 mg). The lowest limit of reliable quantitation was established at 1 ng/ml in plasma. The intra-assay precision (coefficient of variation) remained less than 6% at all concentrations evaluated from 1 to 800 ng/ml. In urine, the lowest limit of quantitation was validated to 20 ng/ml where the intra-assay precision (coefficient of variation) for unchanged drug was less than 4% at all concentrations evaluated up to 400 ng/ml. This method is suitable for routine quantitation of unchanged drug in human plasma and urine following the administration of therapeutically effective doses of dilevalol hydrochloride.     

Sensitive determination of piritramide in human plasma by gas chromatography.

A selective and sensitive method for the determination of piritramide in human plasma is described. A 1-ml aliquot of plasma was extracted with 10 ml of hexane-isoamyl alcohol (99.5:0.5, v/v) (extraction efficiency 86%) after addition of 50 microliters of 2 M ammonia and 20 microliters of aqueous strychnine solution (100 ng per 10 microliters) as internal standard. Gas chromatography was performed with J&W DB-1, 30 m x 0.53 mm I.D. separation column, film thickness 1.5 microns, using an nitrogen-phosphorus-sensitive detector. The assay was linear in the concentration range 3.75-2250 ng/ml (r = 0.999), with a lower limit of detection of 1-2 ng/ml. The precision was determined using spiked plasma samples (10 and 50 ng/ml), with coefficients of variation of 3.5 and 3.1% (intra-day; n = 5) and 4.6 and 4.1% (inter-day; n = 4). In the range 3.75-150 ng/ml, the accuracy of the assay was 3.36%. The method was used for the determination of piritramide plasma concentrations in patients receiving intra- or post-operative analgesia.     

High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.     

Single-drop liquid-phase microextraction for the determination of hypericin, pseudohypericin and hyperforin in biological fluids by high performance liquid chromatography

The analysis of hypericin, pseudohypericin (collectively called in this study hypericins) and hyperforin in biological fluids is reported using single-drop liquid-phase microextraction in conjunction with HPLC-UV-fluorescence detection. A new option for analysis of the active principle constituents in biological samples is proposed, reducing the steps required prior to analysis. There are several parameters which determine the mass transfer such as the extraction solvent, drop and sample volumes, extraction time and temperature, pH and ionic strength, stirring rate and depth of needle tip in the bulk solution. These parameters were chosen to optimize the performance in the current study. The method was validated with respect to precision, accuracy and specificity. The intra-day precision values were below 2.3% for the high concentration level of control samples and 6.2% for the low level. The respective inter-day precision values were calculated to be below 4.4 and 7.1%, respectively, for the two concentration levels. Accuracy of the method, calculated as relative error, ranged from -2.6 to 7.0%. It was demonstrated that as long as the extraction procedure is consistently applied, quantitative analysis is performed accurately and reproducibly in human urine and plasma samples. Limits of quantitation (LOQs) in urine were calculated to be 3, 6 and 12 ng/ml for pseudohypericin, hypericin and hyperforin, respectively. Slightly higher limits were measured in plasma, i.e. 5, 12 and 20 ng/ml, for the respective analytes.     

A highly sensitive LC-tandem MS assay for the measurement in plasma and in urine of salbutamol administered by nebulization during mechanical ventilation in healthy volunteers

The new-generation nebulizers are commonly used for the administration of salbutamol in mechanically ventilated patients. The different modes of administration and new devices have not been compared. We developed a liquid chromatography-tandem mass spectrometry method for the determination of concentrations as low as 0.05 ng/mL of salbutamol, corresponding to the desired plasma concentration after inhalation. Salbutamol quantification was performed by reverse-phase HPLC. Analyte quantification was performed by electrospray ionization-triple quadrupole mass spectrometry using selected reaction monitoring detection ESI in the positive mode. The method was validated over concentrations ranging from 0.05 to 100 ng/mL in plasma and from 0.18 to 135 ng/mL in urine. The method is precise, with mean inter-day coefficient of variation (CV%) within 3.1-8.3% in plasma and 1.3-3.9% in urine, as well as accurate. The proposed method was found to reach the required sensitivity for the evaluation of different nebulizers as well as nebulization modes. The present assay was applied to examine whether salbutamol urine levels, normalized with the creatinine levels, correlated with the plasma concentrations. A suitable, convenient and noninvasive method of monitoring patients receiving salbutamol by mechanical ventilation could be implemented.     

Simultaneous analysis of prednisone, prednisolone and their major hydroxylated metabolites in urine by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the simultaneous determination of prednisone, prednisolone and their major hydroxylated metabolites, viz., 20 beta-hydroxyprednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone, in human urine is presented. The retention times were 6.5, 11.4, 18.1, 24.2, 31.6 and 35.3 min, respectively. The technique employs betamethasone as the internal standard. Samples are extracted with ethyl acetate using a diatomaceous earth extraction column, and the extract was dried and injected onto a silica gel column with ultraviolet detection at 254 nm. The calibration curve is linear within the studied range 50-1500 ng/ml for prednisolone and 50-750 ng ml for the other steroids. The intra-day and inter-day coefficients of variation are less than 10% for prednisone and prednisolone but higher for the metabolites. The assay was used to study the excretion rate profile of each of these steroids in the urine of a normal male subject receiving a 49.3-mg intravenous dose of prednisolone. The results indicate that prednisone, 6 beta-, 20 alpha- and 20 beta-hydroxyprednisolone may be the major unconjugated metabolites of prednisolone while 20 beta-hydroxyprednisone may be a minor metabolite.     

Detection, quantification, and identification of dermorphin in equine plasma and urine by LC–MS/MS for doping control

Dermorphin is a unique opioid peptide that is 30–40 times more potent than morphine. It was misused and went undetected in horse racing until 2011 when intelligence obtained from a few North American race tracks suggested its use. To prevent such misuse, a reliable analytical method became necessary for detection and identification of dermorphin in post-race horse samples. This paper describes the first liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for such a purpose. Equine plasma and urine samples were pre-treated with ethylenediamine tetra-acetic acid and urea prior to solid-phase extraction (SPE) on Oasis MCX cartridges. Resulting eluates were dried under vacuum and analyzed by LC–MS/MS for dermorphin. The matrix effect, SPE efficiency, intra-day and inter-day accuracy and precision, and stability of the analyte were assessed. The limit of detection was 10 pg/mL in plasma and 20 pg/mL in urine, and the limit of confirmation was 20 pg/mL in plasma and 50 pg/mL in urine. Dermorphin in plasma is stable at ambient temperature, but its diastereomer is unstable. With isotopically labeled dermorphin as an internal standard, the quantification range was 20–10,000 pg/mL in plasma and 50–20,000 pg/mL in urine. The intra-day and inter-day accuracy was from 91 % to 100 % for the low, intermediate, and high concentrations. The intra-day and inter-day coefficients of variation were less than 12 %. The method differentiates dermorphin from its diastereomer. This method is very specific for identification of dermorphin in equine plasma and urine, as assessed by BLAST search and targeted SEQUEST search, and by MS/MS spectrum library search. The method has been successfully applied to analysis of samples collected following dermorphin administration to research horses and of official post-race samples.

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

Summary This paper describes a high-performance liquid chromatographic (HPLC) assay method for the determination of trichlormethiazide (TCM) in human plasma and urine. After extraction and separation on an ODS column TCM from plasma was detected by oxidation in an electrochemical detector (ECD) by a porous graphite electrode. The sensitivity was better than HPLC with UV detection, enabling the determination of 2 ng ml^–1 TCM in human plasma. This method also allows determination of TCM at higher concentrations by exchanging the UV for the electrochemical detector. To study the pharmacokinetics, TCM in plasma and urine was assayed with coefficients of variation in the range 2–3%. The method has the advantages of high sensitivity for plasma assay and high precision with a simple procedure for both plasma and urine samples. Small samples of 0.5 ml plasma per assay also reduced the total volume of plasma needed.     

Enantiospecific high-performance liquid chromatographic assay with fluorescence detection for the monoamine oxidase inhibitor tranylcypromine and its applicability in pharmacokinetic studies.

In order to be able to measure low concentrations of tranylcypromine enantiomers in biological material, chiral fluorescent derivatization and high-performance liquid chromatography (HPLC) were employed. The internal standard S-(+)-amphetamine and borate-sodium hydroxide buffer pH 11 were added to plasma or urine sample aliquots. o-Phthaldialdehyde was used for precolumn derivatization in combination with the chiral mercaptan N-acetylcysteine. HPLC resolution of the diastereoisomeric derivatives was possible on an octadecylsilane column. The mobile phase consisted of sodium phosphate buffer solution pH 6.5, methanol and tetrahydrofuran. The fluorescence of the eluate was monitored at 344/442 nm. The intra-day coefficients of variation were below 10%, the limit of determination was 0.5 ng/ml. The assay was found to be applicable for routine analyses in a preliminary pharmacokinetic study, in which an oral dose of 20 mg racemic tranylcypromine sulfate was administered to three healthy volunteers. The plasma concentrations were generally low, and those of S-(-)-tranylcypromine significantly exceeded those of the R-(+)-enantiomer. Average maximum concentrations were 57.5 and 6.3 ng/ml for S- and R-tranylcypromine, respectively. While S-tranylcypromine was well detectable within the whole study period (8 h), R-tranylcypromine concentrations fell below the detection limit after 4 h in two out of the three studied volunteers.     

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.     

Simple and extractionless high-performance liquid chromatographic determination of rosiglitazone in human plasma and application to pharmacokinetics in humans

A simple and extractionless HPLC method using fluorescence detection was developed for the determination of rosiglitazone in human plasma. After deproteinization using perchloric acid the plasma samples were directly injected onto the HPLC system. The mobile phase was composed of acetonitrile (52%) and 20 mm ammonium acetate (48%, pH 7.5), and analysis was run at a flow rate of 0.2 mL/min with the detector operating at 247 nm for excitation wavelength and at 367 nm for emission wavelength, respectively. The method has a mean recovery of 97%, while the intra-day and inter-day precisions were all less than 7%. This method is simple, specific, sensitive and requires only a small plasma volume with short analytical time, and is suitable for the determination of plasma rosiglitazone in routine measurements for pharmacokinetic studies.     

Determination of Saccharin in Plasma and Urine by High Performance Liquid Chromatography

Abstract

A sensitive and specific high performance liquid chromatographic (HPLC) assay for the determination of saccharin in plasma and urine was developed. Saccharin is extracted into diethyl ether at acid pH, evaporated, and reconstituted prior to instrumental analysis. Overall recovery of saccharin is 86.9 + 8.6% and the sensitivity limits of detection is 0.15 μg per ml of plasma or urine using a fluorescence detector. The sensitivity limit in plasma can be extended to 20 ng per ml by use of a 2 ml assay volume and detector attenuation. The assay was used for the determination of saccharin in plasma and urine of rats following oral doses of 5 mg/kg.     

Determination of YM992, a novel selective serotonin reuptake inhibitor, in rat and dog plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method with fluorescence detection was developed for the determination of (S)-2-[[(7-fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride (YM992) in plasma. Plasma samples were extracted with n-hexane under alkali condition. After the organic solvent was evaporated to dryness, the residue was treated with 4-fluoro-7-nitrobezofurazan (NBD-F) in borate buffer (pH 7.5) at room temperature for 20 min. The reaction was terminated with hydrochloric acid and the resultant solution was injected onto HPLC without further purification. No interfering peak was observed at the retention time of YM992 or the internal standard. The calibration curve was linear with the concentration of YM992 up to 200 ng/ml. The limit of quantitation was 1 ng/ml. The intra- and inter-day relative standard deviation was less than 5.6% and 4.1%, respectively, and the intra- and inter-day relative error ranged from -3.0% to 17.2% and 2.8% to 7.5%, respectively. Using the assay, the plasma concentration of YM992 could be determined up to 8 and 10 h after the oral administration of YM992 to rats and dogs, respectively.     

Quantitation of pancuronium, 3-desacetylpancuronium, vecuronium, 3-desacetylvecuronium, pipecuronium and 3-desacetylpipecuronium in biological fluids by capillary gas chromatography using nitrogen-sensitive detection

A sensitive and specific capillary gas chromatographic (GC) assay was developed for the quantitation of the quaternary ammonium steroidal neuromuscular blocking drugs pancuronium (PANC), vecuronium (VEC) and pipecuronium (PIP), as well as the metabolites 3-desacetylpancuronium (3-desPANC) and 3-desacetylvecuronium (3-des VEC) in plasma, bile and urine; the putative metabolite 3-desacetylpipecuronium (3-des PIP) was extracted and quantitated only in urine. The procedure employed a single dichloromethane extraction of the iodide ion-pairs of the monoquaternary or bisquaternary ammonium compounds (including internal and external standards) from acidified, ether-washed biological fluid followed by the formation of stable O-tert.-butyldimethylsilyl derivatives at the 3-hydroxy steroidal position of the metabolites. An automated capillary GC system fitted with a nitrogen-sensitive detector and an integrator was then used to analyze and quantitate both parent compounds and their derivatized metabolites. Optimal extraction, derivatization and GC conditions, as well as short-term stability and recoveries of these drugs and metabolites in plasma, are reported. Electron ionization mass spectrometry combined with GC was used to confirm the identities of compounds eluted from the column. The assay demonstrated a 10(3)-fold linear range up to 5000 ng/ml for PANC, VEC, 3-des VEC and PIP, and lower limits of detection with adequate precision of 2 ng/ml for PANC, VEC and PIP, and 4 ng/ml for 3-des VEC; 3-des PANC was linear from 8 to 500 ng/ml while 3-des PIP was linear from 25 to 1000 ng/ml. The precision (coefficient of variation) of the calibration curves for underivatized drugs and their derivatized metabolites over the linear ranges was 2-20% and the reproducibility of the assay over a range of clinical concentrations of these drugs found in human plasma was 5-16% for PANC, 2-4% for VEC and 6-11% for PIP. No interferences were detected in the assay of plasma samples from 106 surgical patients.     

使用限进介质色谱柱的柱切换高效液相色谱法直接进样测定大鼠血浆中舒必利

提出一种直接进样测定大鼠血浆中舒必利浓度的高效液相色谱方法,使用限进介质色谱柱作为预柱在线去除血浆蛋白后,将舒必利通过柱切换转移到分析柱中进行分析。限进介质色谱柱为CAPCELLPAKMFSCX阳离子交换柱（20&#215;4．0mmi．d．,5μm）,分析柱为Kromasil C18柱（150&#215;4．6mm i．d．,5μm）,限进介质柱预分离时流动相为PH=6．88的50mmol／L磷酸盐缓冲液乙腈（100：5,V／V）,切换后分析流动相为PH=6．83的50mmol／L磷酸盐缓冲液-乙腈（100：10,V／V）。流速均为1mL／min,检测波长为240nm。该方法检出限为17ng／mL,定量限为50ng／mL。舒必利在50～1400ng／mL之间线性良好（r=0．9997）,高中低浓度的日内、日间相对标准偏差分别为1．5％～4．2％及2．0％～5．2％,方法回收率为98．8％～104．1％．     

High-performance liquid chromatographic evaluation of 2-(alpha-thenoylthio)propionylglycine and its two metabolites in biological fluids

A high-performance liquid chromatographic (HPLC) method for determining 2-(alpha-thenoylthio)propionylglycine (TTPG) and its two main metabolites, thiophenecarboxylic acid and thiopronine, in biological samples was developed. TTPG and its metabolites were extracted by solvent partition and then determined by reversed-phase HPLC with UV detection at 245, 295 and 360 nm. This procedure was validated in order to allow the assay of these compounds in plasma and urine samples with sufficiently low detection limits (50 ng/ml for TTPG and TCA and 100 ng/ml for thiopronine) and with good linearity within the concentration range investigated. It was applied to a comprehensive pharmacokinetic investigation of TTPG in healthy volunteers.