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.     

Determination of sparfloxacin in serum and urine by high-performance liquid chromatography.

A specific and sensitive analytical method for the determination of sparfloxacin in serum and urine is described. Serum proteins are removed by precipitation with acetonitrile after the addition of ofloxacin as an internal standard. The supernatant solvent is evaporated in a vacuum concentrator and the dry residue is redissolved in the mobile phase. Separation is performed on a cation-exchange column (Nucleosil 100 5SA, 125 x 4.0 mm I.D., 5 microns particle size) protected by a guard column (Perisorb RP-18, 30 x 4.0 mm I.D., 30-40 microns particle diameter). The mobile phase consisted of 750 ml of acetonitrile and 250 ml of 100 mmol/l phosphoric acid (v/v) to which sodium hydroxide had been added. The final concentration of sodium was 23 mmol/l and the pH was 3.82. Sparfloxacin and ofloxacin were determined by spectrofluorimetry (excitation wavelength 295 nm; emission wavelength 525 nm). The flow-rate was 1.5 ml/min and the retention times were 4.7 (sparfloxacin) and 8.0 (ofloxacin) min. Validation of the method yielded the following results for serum: detection limit 0.05 mg/l; precision between series 10.4-3.6%; recovery 99.5-100.0%; comparison with a microbiological assay c(bioassay) = 1.035c(HPLC) - 0.06. The test organism was Bacillus subtilis ATCC 6633. For urine the results were: detection limit 0.5 mg/l; precision between series 7.8-5.0%; recovery 97.0-97.8%; method comparison c(bioassay) = 1.092c(HPLC) - 1.09. No interferences were observed in human volunteers. The method can also be applied to stool samples.     

Determination of amitriptyline and nortriptyline in human plasma by quantitative thin-layer chromatography

A thin-layer chromatographic method for simultaneous determination of amitriptyline (AT) and nortriptyline (NT) in human plasma is described. Both substances are extracted from biological material by means of a single extraction. The extract is evaporated until dry and the residue quantitatively applied to a silica gel thin-layer plate. AT and NT are separated from interfering plasma components by chromatography. The spots are visualized by nitration, reduction and coupling with N-(1-naphtyl)ethylenediamine on the plate. The intensity of the azo-dyes formed can be measured densitometrically. Using 1 ml of plasma, the sensitivity limit was 0.5 ng/ml for both substances. About 10--15 plasma samples can be analysed per day. The method is applicable to pharmacokinetic studies after a single oral dose of 25 mg AT as hydrochloride in man.     

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.     

Determination of fluorescent trypanocidal diamidines by quantitative thin-layer chromatography

The fluorescent trypanocidal diamidines 2-(4-amidinophenyl)indole-6-carboxamidine dihydrochloride (I, DAPI), 2-(4-amidinophenyl)benzo[b]thiophene-6-carboxamidine dihydrochloride (II) and 2-(4-amidinophenyl)-1-benzofurane-5-carboxamidine dihydrochloride (III) were determined in plasma, urine, faeces and tissues of experimental animals using quantitative thin-layer chromatography. Samples were extracted with n-octanol after addition of sodium hydroxide and subsequently re-extracted into 0.1 M hydrochloric acid. Chromatography was performed on silica gel plates under nitrogen with n-butanol saturated with 2 M hydrochloric acid. Quantitation was performed by measuring native fluorescence using a fluorodensitometer. The respective diamidines were used as internal standards for each other to ensure precision (coefficient of variation less than 7%) and accuracy of the assay. Calibration curves were linear up to 150 ng/ml of sample solution with detection limits of 10 ng/ml of sample solution for I and III and 50 ng/ml for II. The described method has been successfully used for pharmacokinetic studies in experimental animals.     

Determination of (15R)- and (15S)-15-methylprostaglandin E2 in human plasma with picogram per milliliter sensitivity by column-switching high-performance liquid chromatography

(15R)-15-Methylprostaglandin E2 (PGE2) is a pro-drug under evaluation for the treatment of acute upper gastrointestinal hemorrhage and gastrointestinal cytoprotection. It is converted in acid (e.g., gastric fluid) to its active 15S epimer. Both epimers are found in human plasma at low pg/ml levels following oral dosing. A high-performance liquid chromatographic (HPLC) method was developed for the simultaneous analysis of (15R)- and (15S)-15-methyl-PGE2 in human plasma. The method combined off-line solid-phase extraction and reversed-phase HPLC clean-up with panacyl bromide derivatization and subsequent analysis using a heteromodal column-switching technique. Assay linearity was demonstrated over a range of 10-200 pg/ml for both 15-methyl-PGE2 epimers (r greater than or equal to 0.995). There were no significant inter-day differences in assay results for either epimer at 50 and 25 pg/ml (p greater than 0.05), with pooled estimates of precision at these levels producing relative standard deviations of less than or equal to 8% and less than or equal to 12%, respectively. The method quantitation limit (signal-to-noise ratio 5:1) for both epimers was 10 pg/ml when processing 3 ml of plasma. The analysis procedure was shown to be useful for quantifying at or below 10% of the (15R)-15-methyl-PGE2 maximum plasma concentration following a 50-micrograms oral dose in three human volunteers. For the same three subjects, however, the plasma concentration of (15S)-15-methyl-PGE2 did not exceed the quantitation limit of 10 pg/ml.     

Determination of ofloxacin in human serum by high-performance liquid chromatography with column switching.

The chromatographic behaviour of ofloxacin on various sorbents, including ODS, C8, C1, nitril, phenyl and tert,-butyl, as stationary phases was investigated and a high-performance liquid chromatography (HPLC) assay was developed for the determination of ofloxacin in serum. The serum samples were directly introduced onto an HPLC column after filtering through a Morcut II membrane filter to remove proteins. The filtrate was concentrated on a pre-column using a phenyl stationary phase and was then introduced to an analytical column with an ODS stationary phase by column switching. Ofloxacin and enoxacin as an internal standard were detected by ultraviolet absorbance at 300 nm. Determination was possible for ofloxacin over the concentration range 50-2000 ng/ml; the limit of detection was 20 ng/ml. The recovery of ofloxacin added to serum was 88.8-101.7% with a coefficient of variation of less than 5.2%. This method is applicable to pharmacokinetic studies of patients after treatment with ofloxacin.     

Determination of diazepam and nordazepam in milk and plasma in the presence of oxazepam and temazepam

For studies on the excretion of drugs into milk a sensitive high-performance liquid chromatographic assay was developed to quantitate diazepam and nordazepam in the milk and plasma of humans and rabbits in the presence of their major metabolites, oxazepam and temazepam. Flurazepam was used as an internal standard. The assay involves extractions with diethyl ether and an additional acid clean-up step. Chromatographic separation was achieved by a LiChrospher 60 RP-select B (5 microns) column and KH2PO4- acetonitrile (69:31, v/v) adjusted to pH 2.80 as a mobile phase. The same extraction and chromatographic conditions were suited to both types of samples, milk and plasma. The limits of determination using ultraviolet detection at 241 nm was for diazepam 20 ng/ml and for nordazepam 15 ng/ml. The absolute recoveries of diazepam, nordazepam and flurazepam in human milk were 84, 86 and 92% and in human plasma 97, 89 and 94%, respectively. The within- and between-day accuracy and precision for diazepam and nordazepam in milk and plasma at all concentrations tested (20-1500 ng/ml) were better than 8%. The high fat content which occurs in rabbit milk presented no limitation for the extraction of lipophilic diazepam: the method was successfully used to monitor milk and plasma concentrations of diazepam and nordazepam in lactating New Zealand White rabbits during 26-h infusions of diazepam (1.4 mg/h).     

Determination of tazobactam and piperacillin in human plasma, serum, bile and urine by gradient elution reversed-phase high-performance liquid chromatography

A gradient elution high-performance liquid chromatographic method is described for the analysis of the beta-lactamase inhibitor tazobactam (YTR-830H) and a semi-synthetic parenteral penicillin, piperacillin, in human plasma, serum, bile and urine. The assay for plasma, serum and bile involves deproteinization with acetonitrile and the removal of lipids with dichloromethane; urine is diluted with buffer. Separation and quantitation are achieved using a mobile phase based on ion-suppression chromatography on a C18 reversed-phase column with ultraviolet detection at 220 nm. The limit of quantitation for both compounds is 1.0 microgram/ml in plasma, serum and bile using a 0.2-ml sample and 50.0 micrograms/ml in urine using a 0.1-ml sample. The method has been validated by preparing and analyzing a series of fortified samples (range 1.0-200 micrograms/ml for each compound in plasma, serum and bile and 50.0-10,000 micrograms/ml for each compound in urine). Excellent linearity, accuracy, precision and recovery were obtained. The method was not interfered with by other endogenous components, nor by other commonly administered antibiotics such as amoxicillin, mezlocillin, cefometazole and cefotaxime. The assay has been successfully applied to the analysis of samples from pharmacokinetic studies in man and animals.     

Quantitative determination of SC-68328 in dog plasma using flow injection and tandem mass spectrometry

A flow injection/tandem mass spectrometric assay was developed to quantitate SC-68328 in dog plasma using its stable isotopic analog [13C4]SC-68328 as an internal standard (IS). Since SC-68328, a manganese-based superoxide dismutase mimetic, is very unstable, very polar and adheres to silica-based high-performance liquid chromatographic columns, the analyte and IS were derivatized to their bis-isothiocyanate forms followed by a liquid-liquid extraction with methylene chloride and analyzed using positive ion electrospray mass spectrometric detection. SC-68328 was quantitated using the peak-height ratio of SC-68328 to its IS using MS/MS in the multiple reaction monitoring mode. The lower limit of quantitation of the assay was 0.25 microg ml(-1) SC-68328 in dog plasma with an inter-day precision of 11.8% and an accuracy of 113% (n = 12). Acceptable precision and accuracy were also obtained for concentrations in the calibration curve range (0.25-10 microg ml(-1) SC-68328 in dog plasma).     

Quantitation of the enantiomers of ofloxacin by capillary electrophoresis in the parts per billion concentration range for in vitro drug absorption studies

Ofloxacin, a chiral fluoroquinolone, possesses two optical isomers. The antibacterial activity of S-(-)-ofloxacin is reported to be 8-128 times higher than that of R-(+)-ofloxacin. A capillary zone electrophoresis method has been developed to quantify the enantiomers of ofloxacin in high diluted samples (20-700 ng/ml for each enantiomer). After fluid-fluid extraction of ofloxacin from physiological solution electrokinetic injection was employed to improve the sensitivity. The method was optimised using a central composite design. Four experimental factors were investigated: the background electrolyte concentration, the methyl-beta-cyclodextrin concentration, the buffer pH and the temperature. The amount migrated into the capillary, determined by the peak area, the resolution between the ofloxacin enantiomers, the migration time and the generated current were evaluated as responses. The quantification limit is 11.4 ng/ml for S-ofloxacin and 10.8 ng/ml for R-ofloxacin. The method has shown good validation data in terms of precision and recovery rate.     

Combined thin-layer chromatography-photography-densitometry for the quantification of ifosfamide and its principal metabolites in urine, cerebrospinal fluid and plasma.

A method has been devised for the determination of the anticancer drug ifosfamide and its principal metabolites in urine, plasma and cerebrospinal fluid (CSF). The urine and CSF samples are absorbed onto Amberlite XAD-2 eluting the compounds of interest with methanol. Plasma is deproteinated using cold acetonitrile and centrifuged to yield a clear supernatant. The eluate and supernatant are analyzed by thin-layer chromatography, with spot visualization using 4-(4-nitrobenzyl)pyridine. The plates are photographed for subsequent densitometeric analysis. The intra-assay coefficient of variation for each compound in both urine and plasma was less than 10% and the lower limit of detection was 1 microgram/ml. The method provides a means of determining the full spectrum of metabolic products of ifosfamide in patients and will allow detailed investigation of variability in metabolism and pharmacokinetics of this drug.     

Determination of cefquinome in pig plasma and bronchoalveolar lavage fluid by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5-2500 ng ml(-1) for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4-1000 ng ml(-1). The limit of quantification (LOQ) was set at 5.00 ng ml(-1) for plasma and at 4.00 ng ml(-1) for BAL fluid. The limit of detection (LOD) was 3.12 ng ml(-1) and 0.41 ng ml(-1) for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml(-1) for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied.     

High-performance liquid chromatographic determination of plasma and urinary 1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3,7-dicarboxylic acid.

A high-performance liquid chromatographic method for the analysis of 1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3,7-dicarboxylic acid (I) in plasma and urine is described. A statistical evaluation of the assay technique has shown acceptable accuracy and precision at concentrations as high as 2.0 microgram/ml of plasma or 29.0 microgram/ml of urine for samples augmented with 1. As little as 0.08 microgram/ml of I in plasma or 0.42 microgram/ml of I in urine were quantitatively determined. The mean relative error for the assay of unknown concentrations of I in plasma and urine was +/- 8% and +/- 3%, respectively. This method was used for the analysis of I in the plasma and urine of rhesus monkeys following oral administration of 200 mg/kg of nalidixic acid.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography.

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane-ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile-ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5-500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63-74 and 63-68%, respectively, over a concentration range of 0.5-500 ng/ml.     

High-performance liquid chromatographic assay for mitoxantrone in plasma using electrochemical detection

A sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of mitoxantrone in plasma using electrochemical detection. Bisantrene was chosen as the internal standard. A reversed-phase, 10-microns muBondapak C18 analytical column (30 cm X 3.9 mm) with an isocratic mobile phase of 28% acetonitrile in 80 mM sodium formate buffer (pH 3.0) was used. The eluent was monitored by both electrochemical detection at an applied potential of +0.75 V vs. Ag/AgCl and visible absorbance at 660 nm. Only electrochemical detection was able to quantitate the internal standard and provided ten times higher sensitivity than visible absorbance for mitoxantrone with a detection limit as low as 0.1 ng/ml. Calibration curves in the range 0.1-1000 ng/ml showed good linearity (r = 0.998) and precision (coefficient of variation less than 10%). This HPLC method utilized a reproducible and inexpensive liquid-liquid extraction procedure. Using methylene chloride, the extraction efficacy of mitoxantrone from plasma was 85.3% with a coefficient of variation less than 2.1%. This new assay was then applied to measure mitoxantrone concentrations in plasma obtained from two leukemic patients receiving 12 mg/m2 mitoxantrone as a 1-h infusion.     

High Pressure Liquid Chromatography Assay for Determination of 18-β-Glycyrrhetinic Acid in Plasma

Abstract

This report presents a useful high pressure chromatography assay for determination of the proposed chemopreventive agent 18-β-glycerrhetinic acid (GA) in murine and human plasma. Drug was released from plasma proteins through precipitation with a mixture of sodium bisulfate and sodium chloride after which it was extracted with acetonitrile. Standard calibration curves of GA covered the concentration range of 2.5 to 120 μg/ml. The lower limit of detection was 0.5 μg/ml. No endogenous plasma constituents from plasma were found to interfere with the determination of GA. Recover of GA from plasma was greater than 95% over a concentration range of 20 to 100 μg/ml. Linearity of the assay was excellent; within-run precision showed a C.V. of 4.2% at 25 μg/ml, 5.6% at 20 μg/ml and 3.4% at 120 μg/ml. Between-run assay precision and accuracy was also considered to be excellent. The specificity, sensitivity and reproducibility of the procedure are adequate for proposed clinical pharmacology studies of this agent.     

Determination of Ritodrine in Plasma Using HPLC

Abstract

An HPLC instrument coupled with an electrochemical detector was used to determine ritodrine (erythro-p-hydroxy-α-[1-[(p-hydroxyphenethyl)-amino]ethyl] benzyl alcohol hydrochloride) at nanogram levels in serum. Extraction of ritodrine was accomplished using a multistep ethyl acetate procedure, and the mobile phase consisted of acetonitrile, ammonium acetate, glacial acetic acid, and a counterion. The stationary phase was a Biophase ODS 5 μm column at ambient temperature. Nalbuphine hydrochloride (Nubain®) was used as an internal standard to quantitate the ritodrine levels of pregnant patients receiving ritodrine. The procedure's linearity for both ritodrine standards and spiked plasma samples was demonstrated. The precision of the assay was found to be 3.4% at 20 ng/ml ritodrine. The minimum detectable concentration, with a signal-to-noise ratio of 6, was determined to be 0.31 ng per 50 μl injected, corresponding to a concentration of 0.6 ng/ml plasma. The sensitivity, precision, and reproducibility of the assay were all found to be acceptable for determining ritodrine in patient serum.     

Enantioanalysis of bisoprolol in human plasma with a macrocyclic antibiotic HPLC chiral column using fluorescence detection and solid phase extraction

A sensitive, enantioselective, high-performance liquid chromatographic (HPLC) method was developed and validated to determine S-(-)- and R-(+)-bisoprolol in human plasma. Baseline resolution was achieved using the teicoplanin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic T with a polar ionic mobile phase (PIM) consisting of methanol-glacial acetic acid-triethylamine (100 : 0.02 : 0.025, v/v/v) at a flow rate of 1.5 ml/min and fluorescence detection set at 275 nm for excitation and 305 nm for emission. All analyses with S-(-)-atenolol as the internal standard were conducted at ambient temperature. The assay involved the use of a solid-phase extraction procedure for human plasma samples prior to HPLC analysis. The C18 cartridge gave good recovery rates for both enantiomers without any interference. The method was validated over the range of 20-200 ng/ml for each enantiomer concentration. Recovery rates for S-(-)- and R-(+)-bisoprolol enantiomers were in the range of 95-102%. The method proved to be precise (within-run precision expressed as % RSD ranged from 1.0-6.2% and between-run precision ranged from 0.9-6.7%) and accurate (within-run accuracies expressed as percentage error ranged from 0.2-4.8% and between-run accuracies ranged from 0.3-1.7%). The limit of quantitation and limit of detection for each enantiomer in human plasma were 20 and 5 ng/ml, respectively.     

A sensitive and selective liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of actinomycin-D and vincristine in children with cancer

We describe a selective and a highly sensitive assay for actinomycin-D (Act-D) and vincristine (VCR) in plasma employing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) detection. The intraday precision (as defined by the coefficient of variation, CV) based on the standard deviation of replicates of quality control samples ranged from 4.9 to 7.5% and 6.5 to 11.3% with accuracy ranging from 90.7 to 98.1% and 91.2 to 103% for Act-D and VCR, respectively. The interday precision ranged from 7.2 to 10.0% and 11.3 to 13.0% and the accuracy ranged from 94.3 to 102% and 90.7 to 91.6% for Act-D and VCR, respectively. Stability studies showed that Act-D and VCR were stable both during the assay procedure and long-term storage. The lower limit of quantitation (LLOQ) for both Act-D and VCR was 0.05 ng/ml. The analytical method showed excellent sensitivity, precision, and accuracy. This method is robust and is being successfully employed in a pharmacokinetic study of these agents in children with cancer, and is expected to support several ongoing and future pediatric trials.