Determination of lefucoxib in rat plasma, urine, and feces by high-performance liquid chromatography with fluorescence detection: application in pharmacokinetic studies

A sensitive, specific, and reproducible high-performance liquid chromatography (HPLC) method with fluorescence detection was developed for determination of lefucoxib in rat plasma, urine, and feces. The method involved liquid-liquid extraction using methyl tert-butyl ether, and celecoxib was used as the internal standard. The chromatographic separation was performed on a Kromasil C18 column (250.0 mm x 4.6 mm, 5.0 microm) with a mobile phase gradient consisting of water and methanol at a flow rate of 1 ml min(-1). The assay was linear in the range of 5.0-1000.0 ng ml(-1) with a correlation coefficient (r) of 0.9994. The limit of quantification was 5.0 ng ml(-1). Inter- and intra-assay precisions were 相似文献   

Development and comparison of high-performance liquid chromatographic methods with tandem mass spectrometric and ultraviolet absorbance detection for the determination of cyclobenzaprine in human plasma and urine

Sensitive assays for the determination of cyclobenzaprine (I) in human plasma and urine were developed utilizing high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS-MS) and ultraviolet (UV) absorbance detections. These two analytical techniques were evaluated for reliability and sensitivity, and applied to support pharmacokinetic studies. Both methods employed a liquid-liquid extraction of the compound from basified biological sample. The organic extract was evaporated to dryness ,the residue was reconstituted in the mobile phase and injected onto the HPLC system. The HPLC assay with MS-MS detection was performed on a PE Sciex API III tandem mass spectrometer using the heated nebulizer interface. Multiple reaction monitoring using the parent → daughter ion combinations of m/z 276 → 215 and 296 → 208 was used to quantitate I and internal standard (II), respectively. The HPLC-MS-MS and HPLC-UV assays were validated in human plasma in the concentration range 0.1–50 ng/ml and 0.5–50 ng/ml, respectively. In urine, both methods were validatedin the concentration range 10–1000 ng/ml. The precision of the assays, as expressed as coefficients of variation (C.V.) was less than 10% over the entire concentration range, with adequate assay specificity and accuracy. In addition to better sensitivity, the HPLC-MS-MS assay was more efficient and allowed analysis of more biological fluid samples in a single working day than the HPLC-UV method.     

Quantitative determination of Astragaloside IV, a natural product with cardioprotective activity, in plasma, urine and other biological samples by HPLC coupled with tandem mass spectrometry

Astragaloside IV is a novel cardioprotective agent extracted from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. This agent is being developed for treatment for cardiovascular disease. Further development of Astragaloside IV will require detailed pharmacokinetic studies in preclinical animal models. Therefore, we established a sensitive and accurate high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) quantitative detection method for measurement of Astragaloside IV levels in plasma, urine as well as other biological samples including bile fluid, feces and various tissues. Extraction of Astragaloside IV from plasma and other biological samples was performed by Waters OASIS(trade mark) solid phase extraction column by washing with water and eluting with methanol, respectively. An aliquot of extracted residues was injected into LC/MS/MS system with separation by a Cosmosil C18 5 microm, 150 mm x 2.0 mm) column. Acetonitrile:water containing 5 microM NaAc (40:60, v/v) was used as a mobile phase. The eluted compounds were detected by tandem mass spectrometry. The average extraction recoveries were greater than 89% for Astragaloside IV and digoxin from plasma, while extraction recovery of Astragaloside IV and digoxin from tissues, bile fluid, urine and fece ranged from 61 to 85%, respectively. Good linearity (R2>0.9999) was observed throughout the range of 10-5000 ng/ml in 0.5 ml rat plasma and 5-5000 ng/ml in 0.5 ml dog plasma. In addition, good linearity (R2>0.9999) was also observed in urine, bile fluid, feces samples and various tissue samples. The overall accuracy of this method was 93-110% for both rat plasma and dog plasma. Intra-assay and inter-assay variabilities were less than 15.03% in plasma. The lowest quantitation limit of Astragaloside IV was 10 ng/ml in 0.5 ml rat plasma and 5 ng/ml in 0.5 ml dog plasma, respectively. Practical utility of this new LC/MS/MS method was confirmed in pilot pharmacokinetic studies in both rats and dogs following intravenous administration.     

Sensitive liquid chromatography assay with ultraviolet detection for a new phosphodiesterase V inhibitor, DA-8159, in human plasma and urine

A sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection (292 nm) was developed and validated for the determination of the new phosphodiesterase V inhibitor, DA-8159 (DA), in human plasma and urine. A single step liquid-liquid extraction procedure using ethyl ether was performed to recover DA and the internal standard (sildenafil citrate) from 1.0 ml of biological matrices combined with 200 microl of 0.1M sodium carbonate buffer. A Capcell Pak C18 UG120 column (150 mm x 4.6 mm I.D., 5 microm) was used as a stationary phase and the mobile phase consisted of 30% acetonitrile and 70% 20mM potassium phosphate buffer (pH 4.5) at a flow rate of 1.0 ml/min. The lower limit for quantification was 5 ng/ml for plasma and 10 ng/ml for urine samples. Within- and between-run accuracy and precision were < or =15 and < or =10%, respectively, in both plasma and urine samples. The recovery of DA from human plasma and urine was greater than 70%. Separate stability studies showed that DA is stable under the conditions of analysis. This validated assay was used for the pharmacokinetic analysis of DA during a phase I, rising dose study.     

Determination of mycophenolic acid and its phenol glucuronide metabolite in human plasma and urine by high-performance liquid chromatography

Simultaneous determination of mycophenolic acid (MPA) and mycophenolate phenol glucuronide (MPAG) in plasma and urine was accomplished by isocratic HPLC with UV detection. Plasma was simply deproteinated with acetonitrile and concentrated, whereas urine was diluted prior to analysis. Linearity was observed from 0.2 to 50 μg/ml for both MPA and MPAG in plasma and from 1 to 50 μg/ml of MPA and 5 to 2000 μg/ml MPAG in urine with extraction recovery from plasma greater than 70%. Detection limits using 0.25 ml plasma were 0.080 and 0.20 μg/ml for MPA and MPAG, respectively. The method is more rapid and simple than previous assays for MPA and MPAG in biological fluids from patients.     

Electromembrane extraction of trace amounts of naltrexone and nalmefene from untreated biological fluids

Nalmefene and naltrexone are used to block the effects of narcotics and alcohol. In the present work, for the first time a microextraction technique was presented to reduce matrix interferences and improve detection limits of the drugs in urine and plasma samples. Electromembrane extraction (EME) followed by high performance liquid chromatography (HPLC) coupled with ultraviolet (UV) detection was optimized and validated for quantification of nalmefene and naltrexone from biological fluids. The membrane consists 85% of 2-nitrophenyl octyl ether (NPOE) and 15% di-(2-ethylhexyl) phosphate (DEHP) immobilized in the pores of a hollow fiber. A 100 V electrical field was applied to make the analytes migrate from sample solution with pH 2.0, through the supported liquid membrane (SLM) into an acidic acceptor solution with pH 1.0 which was located inside the lumen of hollow fiber. Extraction recoveries in the range of 54% and 75% were obtained in different biological matrices which resulted in preconcentration factors in the range of 109-149 and satisfactory repeatability (2.0相似文献   

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 μm) using water–acetonitrile–methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 μg/ml. The limit of quantitation was 0.08 μg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.     

Quantitation of efletirizine in human plasma and urine using automated solid-phase extraction and column-switching high-performance liquid chromatography

A heart-cut column-switching, ion-pair, reversed-phase HPLC system was used for the quantitation of efletirizine (EFZ) in biological fluids. The analyte and an internal standard (I.S.) were extracted from human EDTA plasma by C₁₈ solid-phase extraction (SPE) using a RapidTrace^® workstation. The eluent from the SPE was evaporated, reconstituted and injected onto the HPLC column. Urine samples were diluted and injected directly without the need of extraction. The compounds of interest were separated from most of the extraneous matrix materials by the first C₁₈ column, and switched onto a second C₁₈ column for further separation using a mobile phase of stronger eluting capability. Linearity range was 10–2000 ng ml⁻¹ for plasma and 0.05–10 μg ml⁻¹ for urine. The lower limit of quantitation (LOQ) was 10 ng from 1 ml of plasma, with a signal-to-noise ratio of 15:1. Inter-day precision and bias of quality control samples (QCs) were <5% for plasma and <7% for urine. Selectivity was established against six other antihistamines, three analogs of efletirizine, and on 12 control plasma lots and nine control urine lots. Recovery was 90.0% for EFZ and 89.5% for I.S. from plasma. One hundred samples can be processed in every 2.75 h on a 10-module RapidTrace^® workstation with minimal human attention. Method ruggedness were tested on three brands of SPE and six different lots of one SPE brand. Performance ruggedness was demonstrated by different analysts on multiple HPLC systems. Analyte stability through sample storage, extraction process (benchtop, freeze–thaw, refrigeration after extraction) and chromatography (on-system, reinjection) was established.     

Determination of a new oral cephalosporin,S-1090, in human plasma and urine by direct injection high-performance liquid chromatography with ultraviolet detection and column switching

Direct injection high-performance liquid chromatographic (HPLC) methods with column switching and UV detection were developed for the rapid and accurate determination of S-1090 in human plasma and urine. An internal-surface reversed-phase pre-column and a C₁₈ analytical column were used for the plasma assay. Two pre-columns packed with cyano and phenyl materials and a C₁₈ analytical column were used for the urine assay. The calibration curves for plasma and urine assays were linear in the ranges 0.09–9 μg/ml and 0.5–100 μg/ml of S-1090, respectively. The relative standard deviations for plasma and urine assays were less than 6% with low relative errors. The established HPLC methods were demonstrated to be useful for clinical pharmacokinetic studies after oral administration of S-1090.     

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

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of cibenzoline (Cipralan ^TM) in human plasma and urine. The assay involves the extraction of the compound into benzene from plasma or urine buffered to pH 11 and HPLC analysis of the residue dissolved in acetonitrile---phosphate buffer (0.015 mol/1, pH 6.0) (80:20). A 10-μ ion-exchange (sulfonate) column was used with acetonitrile—phosphate buffer (0.015 mol/1, pH 6.0) (80:20) as the mobile phase. UV detection at 214 nm was used for quantitation with the di-p-methyl analogue of cibenzoline as the internal standard.The recovery of cibenzoline in the assay ranged from 60 to 70% and was validated in human plasma and urine in the concentration range of 10–1000 ng/ml and 50–5000 ng/ml, respectively. A normal-phase HPLC assay was developed for the determination of the imidazole metabolite of cibenzoline. The assays were applied to the determination of plasma and urine concentrations of cibenzoline and trace amounts of its imidazole metabolite following oral administration of cibenzoline succinate to two human subjects.     

Determination of the enantiomeric composition of a new insulin sensitizer in plasma samples from non-clinical and clinical investigations using chiral HPLC with electrospray tandem mass spectrometric detection

Two drug assays were developed and applied to assess the enantiomeric composition of an insulin sensitizer drug in plasma after administration of its racemate to man, and in human and animal plasma and serum samples generated after in vitro experiments. The sample preparation for the assays consisted either of protein precipitation and column-switching, or liquid-liquid extraction and direct injection. Subsequently, both assays employed chiral HPLC coupled to atmospheric pressure ionization mass spectrometry. An interconversion of the racemate to a mixture enriched with the (+)-enantiomer could be confirmed for all species and biological matrices. The individual enantiomers could be quantified in the concentration range 0.5-500 ng/ml, starting with a 100-microl plasma aliquot. Inter- and intra-assay precision and accuracy were in the range 0.1-7.9 and 88.8-106.0%, respectively. Run times of 5 min for a single sample allows the analysis of more than 200 samples overnight.     

Analysis of risperidone and 9-hydroxyrisperidone in human plasma, urine and saliva by MEPS-LC-UV

Risperidone is currently one of the most frequently prescribed atypical antipsychotic drugs; its main active metabolite 9-hydroxyrisperidone contributes significantly to the therapeutic effects observed. An original analytical method is presented for the simultaneous analysis of risperidone and the metabolite in plasma, urine and saliva by high-performance liquid chromatography coupled to an original sample pre-treatment procedure based on micro-extraction by packed sorbent (MEPS). The assays were carried out using a C8 reversed-phase column and a mobile phase composed of 73% (v/v) acidic phosphate buffer (30 mM, pH 3.0) containing 0.23% triethylamine and 27% (v/v) acetonitrile. The UV detector was set at 238 nm and diphenhydramine was used as the internal standard. The sample pre-treatment by MEPS was carried out on a C8 sorbent. The extraction yields values were higher than 92% for risperidone and 90% for 9-hydroxyrisperidone, with RSD for precision always lower than 7.9% for both analytes. Limit of quantification values in the different matrices were 4 ng/mL or lower for risperidone and 6 ng/mL or lower for the metabolite. The method was successfully applied to plasma, urine and saliva samples from psychotic patients undergoing therapy with risperidone, with satisfactory accuracy results (recovery>89%) and no interference from other drugs. Thus, the method seems to be suitable for the therapeutic drug monitoring of schizophrenic patients using the three different biological matrices plasma, urine and saliva.     

The analysis of arildone in plasma,urine and feces by gas—liquid chromatography with electron-capture detection

The analysis of arildone in plasma, urine and feces by gas—liquid chromatography with electron-capture detection is described. O-(2,3,4,5,6-Pentafluorohenzyl)hydroxylamine is the derivatizing agent for the plasma and urine analysis; 3-nitrophenylhydrazine is utilized for fecal analysis. The mean (± S.E.) minimum quantifiable level of arildone was 1.4 (± 0.2) ng/ml in urine, 6.4 (± 0.1) ng/ml in plasma, and 12.6 (± 1.0) ng/g in feces. The chromatographic response was linear in the range of 0 and 10–120 ng/ml for plasma, 0 and 2.5–20 ng/ml for urine and 0 and 25–250 ng/g for feces. The estimated overall precision of the assay was 5.5%, 6.4% and 8.9% in urine, plasma and feces, respectively.     

Assessment of urine and fecal testosterone metabolite excretion in Chinchilla lanigera males

Endemic chinchilla (Chinchilla spp.) populations are nearly extinct in the wild (South America). In captive animals (Chinchilla lanigera and C. brevicaudata), reproduction is characterized by poor fertility and limited by seasonal breeding patterns. Techniques applied for studying male reproductive physiology in these species are often invasive and stressful (i.e. repeated blood sampling for sexual steroids analysis). To evaluate endocrine testicular function, the present experiments were designed to (a) determine the main route of testosterone excretion (14C-testosterone infusion in four males); (b) validate urine and fecal testosterone metabolite measurements (HPLC was used to separate metabolites and immunoreactivity was assessed in all metabolites using a commercial testosterone radioimmunoassay, and parallelism, accuracy and precision tests were conducted to validate the immunoassay); and (c) investigate the biological relevance of the techniques applied (quantification of testosterone metabolite excretion into urine and feces from five males injected with hCG and comparison between 10 males and 10 females). Radiolabelled metabolites of 14C-testosterone were excreted, 84.7+/-4.2 % in urine and 15.2+/-3.9 % in feces. A total of 82.7+/-4.2% of urinary and 45.7+/-13.6% of fecal radioactivity was excreted over the first 24 h period post-infusion (metabolite concentration peaked at 8.2+/-2.5 h and 22.0+/-7.0 h, respectively). Several urinary and fecal androgen metabolites were separated by HPLC but only fecal metabolites were associated with native testosterone; however, there was immunoreactivity in more than one metabolite derived from 14C-testosterone. After hCG administration, an increase in androgen metabolite excretion was observed (p<0.05). Males excreted greater amounts daily of urinary androgen metabolites as compared with females (p<0.05); this difference was not evident in feces. Results of the present study indicate that the procedure used is a reliable and non-invasive method to repeatedly monitor variations in testicular endocrine activity in this species. It can be a useful tool that would help ensure the survival of the wild populations as well as to provide the basis for a more efficient use by the fur industry.     

Methodology for the analysis of fenbendazole and its metabolites in plasma, urine, feces, and tissue homogenates

New methodology for the extraction and analysis of the anthelmintic fenbendazole and its metabolites from plasma, urine, liver homogenates, and feces from several animal species is presented. Quantitation of fenbendazole and its metabolites was conducted by high-pressure liquid chromatography using ultraviolet detection at 290 nm. The combined extraction and analysis procedures give excellent recoveries in all of the different biological matrices examined. High specificity, low limits of detection, and excellent linearity, accuracy, and inter- and intrasample variability were also obtained. The study of fenbendazole pharmacokinetics in vitro and in vivo should be greatly enhanced through the utilization of these methods.     

Simultaneous determination of abacavir and zidovudine from rat tissues using HPLC with ultraviolet detection

A simple high-performance liquid chromatography (HPLC) method has been developed and validated for the simultaneous determination of abacavir and zidovudine (AZT) in rat plasma, amniotic fluid, fetal, and placental tissues. Extraction of abacavir, AZT, and the internal standard, azidouridine (AZDU) in amniotic fluid was carried out by protein precipitation. Extraction from plasma, fetal and placental homogenates was achieved by using a salting out technique. Chromatographic separation was performed using a C8 column (150 mm x 4.6 mm, 5 microm). The mobile phase consisted of 12% acetonitrile in 25 mM sodium phosphate buffer (adjusted to pH 7 with sodium hydroxide) for the fetus, placenta, plasma and amniotic fluid samples at a flow rate of 0.8 mL/min. The method was validated over the range from 0.05 to 50 microg/mL for both abacavir and AZT in the four biological matrices. The absolute recovery of abacavir ranged from 79 to 94%, while AZT recoveries ranged from 79 to 90% in the different biological matrices. The internal standard recovery ranged from 90 to 92%. Acceptable intra- and inter-day assay precision (<10% R.S.D.) and accuracy (<10% error) were observed over 0.05-50 microg/mL for all four matrices.     

Reversed-phase high-performance liquid chromatography method for the simultaneous quantitation of the lactone and carboxylate forms of the novel natural product anticancer agent 10-hydroxycamptothecin in biological fluids and tissues

Camptothecins are indole alkaloids isolated from a Chinese tree, Camptotheca acuminata, and have a wide spectrum of anticancer activity in vitro and in vivo. A novel camptothecin congener 10-hydroxycamptothecin (HCPT) has been shown to be more active and less toxic than camptothecin, and the lactone HCPT is believed to be responsible for its anticancer activity. In the present study, a reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection was developed and validated for the simulataneous analysis of HCPT for lactone form (I) and carboxylate form (II) in plasma, urine and feces and tissues. Biological samples were prepared by a liquid-liquid extraction method using ice-cold methanol-acetonitrile (1:1, v/v). This method was shown to be reproducible and reliable, with intra- and inter-day variations being less than 7%, and accuracy being 94.3%–102.7%. The limits of determination were 2 ng/ml, 2 ng/ml, 2 ng/g, and 10 ng/ml for HCPT forms I and II in rat plasma, urine, feces, and tissues, respectively. The assay was liner over the range 2–2000 ng/ml (r=0.999, P<0.001) with recoveries of greater than 90% for plasma and urine and approximately 70–80% for feces and tissues homogenates through the extraction procedure. This analytic procedure has been successfully applied to a pharmacokinetic study of HCPT in experimental animals and should be useful in the future human studies.     

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.     

Dopamine β-hydroxylase: Determination of half-life and appearance in lymph fluid after IV injection of 125I-DBH into rats

A 4 day half-life of dopamine beta-hydroxylase (DBH) was determined for rats injected IV with ¹²⁵I-rat DBH from the slow exponential component of radioactivity appearing in plasma, urine, feces and combined urine and feces. Half-life estimates for ¹²⁵I-rat DBH injected IV into WKY and SHR animals did not differ from Sprague Dawley (Zivic Miller) rats. Radioactivity declined in parallel in plasma, urine and feces following IV ¹²⁵I-rat DBH administration and each radioactivity falloff curve could be resolved into two components. The slow phase of the decline of radioactivity excreted into urine and feces from which DBH half-life was calculated occurred between 5 and 25 days after ¹²⁵I-rat DBH injection. The early fast phase which is associated with distribution of the exogenous protein in body fluids and tissues continued for approximately the first 140 hr after DBH injection. The distribution characteristics of IV administered active bovine DBH and ¹²⁵I-rat DBH into the lymphatic system were examined. After active bovine DBH or ¹²⁵I-rat DBH was injected IV into rats, active DBH or radioactivity, respectively, appeared in lymph fluid (thoracic duct) within 20 min; reached peak concentrations within 90 min, and thereafter, declined in parallel with the plasma concentration. The concentration of radioactivity in plasma and lymph fluid were found to be unequal at 9 hr but were equivalent 68–75 hrs after IV injection of ¹²⁵I-rat DBH. Based on the amount of active DBH or radioactivity which accumulates in lymph fluid it is clear that'a substantial amount (> 50%) of the DBH in blood circulates through the lymphatic channels. Analysis of parallel experiments with labelled serum albumin indicate that use of these methods to study plasma proteins do provide sensitive measures of biological half-life and lymphatic distribution characteristics. Specifically for DBH, the results of our study suggest that DBH normally circulates in plasma and lymph fluid with a biological half-life of 4 days.     

Liquid chromatographic assay for common sunscreen agents: application to in vivo assessment of skin penetration and systemic absorption in human volunteers

The purpose of the present study was to develop a reverse-phase high-performance liquid chromatographic (HPLC) assay for quantifying four common sunscreen agents, namely 2-hydroxy-4-methoxybenzophenone, 2-ethylhexyl-p-methoxycinnamate, 2-ethylhexylsalicylate (octylsalicylate) and salicylic acid 3,3,5-trimethcyclohexyl ester (homosalate) in a range of biological matrices. This assay was further applied to study the skin penetration and systemic absorption of sunscreen filters after topical application to human volunteers. Separation was achieved utilizing a Symmetry C(18) column with methanol-water as the mobile phase. The assay permits analysis of the sunscreen agents in biological fluids, including bovine serum albumin (BSA) solution, plasma and urine, and in human epidermis. The assay was linear (r2 > 0.99) with minimum detectable limits of 0.8 ng for oxybenzone, 0.3 ng for octylmethoxycinnamate, and 2 ng for homosalate and octylsalicylate. The inter- and intra-day variation for the four sunscreens was less than 3% at the upper end of the linear range and less than 6% at the lower end. Recoveries of sunscreens from plasma, 4% (w/v) BSA solution and epidermal membranes were within the range of 91-104%. Recoveries from urine of the four sunscreens, and oxybenzone with its metabolites were more than 86%. Up to approximately 1% of the applied dose of oxybenzone and its metabolites was detected in the urine. Appreciable amounts were also detected in the stratum corneum through tape stripping. The HPLC assay and extraction procedures developed are sensitive, simple, rapid, accurate and reproducible. Results from the preliminary clinical study demonstrate significant penetration of all sunscreen agents into the skin, and oxybenzone and metabolites across the skin.