Sensitive High-performance Liquid Chromatographic Assay Research Articles

Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay

Context: Doxorubicin (DOX)-loaded folate-targeted poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] nanoparticles [DOX/FA-PEG-P(HB-HO) NPs] were prepared by the W1/O/W2 solvent extraction/evaporation method for applications in cancer treatment. However, the biodistribution, pharmacokinetics, and targeting of the nanoparticles (NPs) have not yet been studied.Objective: The biodistribution, pharmacokinetics, and targeting of DOX/FA-PEG-P(HB-HO) NPs were evaluated in female BALB/c nude mice bearing HeLa tumors.Materials and methods: Three DOX formulations were injected into the tail vein of the mice at a dosage of 5 mg/kg. At each time point, blood and various tissues were collected. All samples were then processed and analyzed by a validated high performance liquid chromatographic (HPLC) method.Results: The t1/2 values of DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p > 0.05) was found in Cmax between the NPs and free DOX. The Tmax values of the two NPs were prolonged from 0.25 to 1 h. The AUC0–t values were 1.55- and 3.05-folds higher than that of free DOX, and MRT increased to 15.99 h for DOX/P(HB-HO) NPs and 25.14 h for DOX/FA-PEG-P(HB-HO) NPs. For DOX/FA-PEG-P(HB-HO) NPs, the DOX content in the tumors were 10.81- and 3.33-times higher than those for free DOX and DOX/P(HB-HO) NPs at 48 h, respectively.Discussion and conclusions: DOX/FA-PEG-P(HB-HO) NPs displayed reduced cardiac toxicity and improved bioavailability. Moreover, the NPs exhibited a significant extent of DOX accumulation in the tumors, thus suggesting that folate-targeted NPs could effectively transport into HeLa tumors with satisfying targeting.

Validation of a high-performance liquid chromatographic ultraviolet detection method for the quantification of vandetanib in rat plasma and its application to pharmacokinetic studies.

To develop a simple and sensitive high-performance liquid chromatography (HPLC) assay with ultraviolet detection method of vandetanib in rat plasma. Samples were extracted with methanol and acetonitrile, evaporated, and then the residue was reconstituted in mobile phase. Vandetanib and the internal standard (I.S.) trazodone hydrochloride were separated with gradient elution (on a C18 Atlantis column using a mobile phase of acetonitrile/0.5% triethylamine, pH 3.0, with a flow rate of 1.0 ml/min), then detected at 341 nm. A linear curve over the concentration range of 80-4000 ng/ml (R² = 0.9998) was obtained. Intra- and inter-assay accuracy ranged from 98.80% to 103.08% and 95.32% to 98.40%, with high precision (R.S.D. % <5%), respectively. The mean absolute recovery was 96.65%. A simple and sensitive HPLC assay with ultraviolet detection method was developed for the determination of vandetanib in rat plasma. This method is sufficient for pharmacokinetic studies of vandetanib in small animals and may be applied to human pharmacokinetic studies.

Pharmacokinetic properties of indinavir in rat: some limitations of noncompartmental analysis

Background: Compartmental as well as noncompartmental analyses are used routinely in pharmacokinetic analysis. Materials and methods: Pharmacokinetic parameters of the anti-HIV agent, indinavir, have been determined in six male rats applying both the compartmental and the noncompartmental analysis. Results and discussion: A very slow declining phase was found in the indinavir plasma concentration profile using an extended sampling time period and applying a sensitive high-performance liquid chromatography assay method. This apparent terminal elimination phase can cause some significant errors when applying noncompartmental kinetic analysis to the data, with mean residence time (MRT) (544.2 ± 123.2 minutes), total systemic clearance (12.0 ± 2.1 mL/min/kg), and steady-state volume of distribution (Vd (ss)) (6.4 ± 1.0 L/kg) being highly different from the results of compartmental kinetic analysis (MRT, Cltotal, and Vd (ss) values of 23.7 ± 5.9 minutes, 35.18 ± 5.1 mL/min/kg, and 0.84 ± 0.28 L/kg, respectively). The parameters estimated by our noncompartmental approach were also significantly different from the results of the same type of data analysis reported in the literature. Conclusion: The differences in parameter estimations, while being a result of the extended plasma sampling period, which is recommended in noncompartmental analysis, support the priority of applying the compartmental analysis approach in the similar cases with some pre-assumptions, mainly ignoring the final apparent terminal elimination phase(s), very deep tissue, which involves very low drug concentrations.

Pharmacokinetic properties of indinavir in rat: some limitations of noncompartmental analysis

Compartmental as well as noncompartmental analyses are used routinely in pharmacokinetic analysis. Pharmacokinetic parameters of the anti-HIV agent, indinavir, have been determined in six male rats applying both the compartmental and the noncompartmental analysis. A very slow declining phase was found in the indinavir plasma concentration profile using an extended sampling time period and applying a sensitive high-performance liquid chromatography assay method. This apparent terminal elimination phase can cause some significant errors when applying noncompartmental kinetic analysis to the data, with mean residence time (MRT) (544.2 +/- 123.2 minutes), total systemic clearance (12.0 +/- 2.1 mL/min/kg), and steady-state volume of distribution (V(d) (ss)) (6.4 +/- 1.0 L/kg) being highly different from the results of compartmental kinetic analysis (MRT, Cl(total), and V(d) (ss) values of 23.7 +/- 5.9 minutes, 35.18 +/- 5.1 mL/min/kg, and 0.84 +/- 0.28 L/kg, respectively). The parameters estimated by our noncompartmental approach were also significantly different from the results of the same type of data analysis reported in the literature. The differences in parameter estimations, while being a result of the extended plasma sampling period, which is recommended in noncompartmental analysis, support the priority of applying the compartmental analysis approach in the similar cases with some pre-assumptions, mainly ignoring the final apparent terminal elimination phase(s), very deep tissue, which involves very low drug concentrations.

Simultaneous determination of acyclovir, ganciclovir, and (R)‐9‐[4‐hydroxy‐2‐(hydroxymethyl)butyl]guanine in human plasma using high‐performance liquid chromatography

Acyclovir, ganciclovir and (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine are active in vitro against the Epstein-Barr virus (EBV) but their in vivo anti-EBV activity is not well understood. We developed a novel, sensitive high-performance liquid chromatography assay with ultraviolet detection for measuring acyclovir, ganciclovir and (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine in human plasma to identify quantitative relationships between in vitro anti-EBV activity and therapeutic response. Characteristics of the assay include a low plasma volume (200 microL), perchloric acid protein precipitation, use of penciclovir as the internal standard, run times less than 8 min and a 50 ng/mL lower limit of quantification. The within- and between-assay variability is 0.7-4.8 and 1.0-7.9%, respectively. Accuracy for all three drugs ranges from 89.5 to 106.4% for four quality controls (50, 100, 1000 and 10,000 ng/mL). This assay supports pharmacokinetic and pharmacodynamic studies of candidate anti-EBV drugs in children and adults with EBV infections.

Simultaneous quantification of cefpirome and cetirizine or levocetirizine in pharmaceutical formulations and human plasma by RP-HPLC

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the simultaneous determination and quantification of cefpirome and cetirizine or cefpirome and levocetirizine in pharmaceutical formulations and human plasma without changing the chromatographic conditions is described. Chromatographic separations were performed on a prepacked Nucleosil 120, C18 (5 μm, 12.5 ± 0.46 mm) column using CH3CN: H2O (75: 25, v/v) as a mobile phase at a flow rate of 1 mL/min while UV detection was performed at 232 nm for monitoring the effluent. A number of other brands of C18 columns were also employed which had a significant effect on the separation. The method has been validated over the concentration range of 0.5–50 μg/mL (r2 > 0.999). The limit of detection (LOD) and quantification (LOQ) for cefpirome and levocetirzine in pharmaceutical formulations and serum were in the range 0.24–1.31 μg/mL. Analytical recovery from human plasma was >98%, and the within and between-day relative standard deviation was <3.1%. The small sample volume and simplicity of preparation make this method suitable for use in pharmaceutical industries, drug research centers, clinical laboratories, and forensic medical centers.

A sensitive and reproducible fluorescent-based HPLC assay to measure the activity of acid as well as neutral β-glucocerebrosidases

The activity of lysosomal acid β-glucocerebrosidase (AGC, EC 3.2.1.45), which hydrolyzes the O-glycosidic linkage between d-glucose and ceramide of glucosylceramide (GlcCer), is a marker for the diagnosis of Gaucher disease because the disease is caused by dysfunction of AGC due to mutations in the gene. The activity of AGC is potently inhibited by conduritol B epoxide (CBE), whereas CBE-insensitive nonlysosomal neutral β-glucocerebrosidase (NGC) activities have been found in various vertebrates, including humans. We report here a new reliable method to determine AGC as well as NGC activities using normal-phase high-performance liquid chromatography (HPLC) and NBD (4-nitrobenzo-2-oxa-1,3-diazole)- or BODIPY (4,4-difluoro-4-bora-3a,4a-diaza- s-indacene)-labeled GlcCer as a substrate. The reaction products of the enzymes, C6-NBD-ceramide and C12-BODIPY-ceramide, were clearly separated from the corresponding substrates on a normal-phase column within 5 min using a different solvent system. Reaction products could be detected quantitatively at concentrations ranging from 50 fmol to 50 pmol for C6-NBD-ceramide and from 10 fmol to 5 pmol for C12-BODIPY-ceramide. V max/ K m values of human fibroblast AGC for fluorescent GlcCer were much higher than those for 4-methylumbelliferyl-β- d-glucoside (4MU-Glc), which is used prevalently for Gaucher disease diagnosis. As a result, AGC activity was detected quantitatively using fluorescent GlcCer, but not 4MU-Glc, using 5 μl of human serum or 1 × 10 4 cultured human fibroblasts. The current method clearly showed the decrease of AGC activities in fibroblasts and serum from the patient with Gaucher disease compared with normal individuals, suggesting that the method is applicable for the diagnosis of Gaucher disease. Furthermore, this method was found to be useful for measuring the activities of nonlysosomal NGC of various cells and tissues in the presence of CBE.

Sensitive fluorescence HPLC assay for AQ-13, a candidate aminoquinoline antimalarial, that also detects chloroquine and N-dealkylated metabolites☆

A sensitive, specific and reproducible fluorescence high performance liquid chromatography (HPLC) assay has been developed for the separate or simultaneous measurement of AQ-13 (a candidate 4-aminoquinoline antimalarial), chloroquine (CQ), and their metabolites in whole blood. After liquid-solid extraction using commercially available extraction cartridges, these two aminoquinolines (AQs) and their metabolites were separated on C18 (Xterra RP18) columns using a mobile phase containing 60% borate buffer (20 mM, pH 9.0) and 40% acetonitrile with isocratic elution at a flow-rate of 1.0 ml/min. The assay uses a biologically inactive 8-chloro-4-aminoquinoline (AQ-18) as its internal standard (IS). There is a linear relationship between the concentrations of these AQs and the peak area ratio (ratio between the peak area of the AQ or metabolite and the peak area of the IS) on the chromatogram. Linear calibration curves with correlation coefficients > or = 0.997 (r2 > or = 0.995, p < 0.001) were obtained for AQ-13, CQ and their N-dealkylated metabolites. Reproducibility of the assay was excellent with coefficients of variation (CVs) < or = 3.8% for AQ-13 and its metabolites, and < or =2.5% for CQ and its metabolites. The sensitivity of the assay is 5 nM using 1.0 ml of blood and a 20 microl injection volume, and can be increased by using 5.0 ml of blood with an injection volume of 40 microl.

Liquid chromatography–tandem mass spectrometry method for determination of phencynonate in rat blood and urine

A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid-liquid extraction with ethyl ether-dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150 mm x 2.1mm i.d., 3 microm). The mobile phase was composed of methanol-water (85:15, v/v), containing 0.5 per thousand formic acid, which was pumped at a flow-rate of 0.2 ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25 min, thiencynonate eluted at approximately 5.10 min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1-100 ng/ml in rat blood and 1-500 ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1 ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57-79.49% in blood and 56.85-64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies.

Comparative bioavailability study of cefixime (equivalent to 100 mg/5 ml) suspension (Winex vs Suprax) in healthy male volunteers

This investigation was carried out to evaluate the bioavailability of a new suspension formulation of cefixime (100 mg/5 ml), Winex, relative to the reference product, Suprax (100 mg/5 ml) suspension. The bio-availability study was carried out in 24 healthy male volunteers who received a single oral dose (200 mg) of the test (A) and the reference (B) products on 2 treatment days after an overnight fast of at least 10 hours. The treatment periods were separated by a one-week washout period. A randomized, balanced two-way crossover design was used. After dosing, serial blood samples were collected over a period of 16 hours. Plasma concentrations of cefixime were analyzed using a sensitive high-performance liquid chromatographic assay. The pharmacokinetic parameters for cefixime were determined using standard non-compartmental method. The parameters AUC(0-t), AUC(0-infinity), Cmax, Kel, t1/2 and Cmax/AUC(0-infinity) were analyzed statistically using raw and log-transformed data. The time to maximum concentration (tmax) was analyzed using raw data. The parametric 90% confidence intervals of the mean values of the pnfinity harmacokinetic parameters: AUC(0-t), AUC(0-infinity) Cmax, and Cmax/AUC(0-infinity) were within the range 80 - 125% which is acceptable for bioequivalence (using log-transformed data). The calculated 90% confidence intervals based on the ANOVA analysis for the mean test/reference ratios of AUC(0-t), AUC(0-infinity), Cmax, and Cmax/AUC(0-infinity) were 88.93 - 107.10%, 89.09 - 107.11%, 89.63 - 108.58% and 96.85 - 105.29%, respectively. The test formulation was found bioequivalent to the reference formulation with regard to AUC(0-t), AUC(0-infinity), and Cmax using the Schuirmann's two one-sided t-tests. Therefore, the two formulations were considered to be bioequivalent.

Sensitive high-performance liquid chromatographic assay for d-amino-acid oxidase activity in mammalian tissues using a fluorescent non-natural substrate, 5-fluoro- d-tryptophan

A sensitive assay for d-amino-acid oxidase (DAO) activity in mammalian tissues has been established. d-Tryptophan ( d-Trp) analogs were tested as substrates for DAO, and 5-fluoro- d-tryptophan ( d-FTP) was found to be the best substrate. By the enzymatic reaction, d-FTP was converted to 5-fluoroindole-3-acetic acid (FIAA), a highly fluorescent product, and the product was determined by an RP-HPLC system with a fluorescence detector. The detection limit for purified DAO (from hog kidney) was 0.25 μU, and the within-day and day-to-day precisions of the assays were 4.6% (RSD, n = 5), and 13.8% (RSD, 5 days), respectively. By the present method, the detailed distribution of DAO activity in the mouse brain was determined using individual animals for the first time, and significant activities were observed in the cerebellum, medulla oblongata and midbrain. Because sensitive DAO assay is frequently required in small tissues or in limited-tissue regions, the present method is useful for various research studies concerning DAO and the related d-amino acids.

High-performance liquid chromatographic assay for the determination of total and free topotecan in the presence and absence of anti-topotecan antibodies in mouse plasma

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay has been developed to allow determination of total (i.e. bound and unbound) and free (i.e. unbound) topotecan (TPT) in mouse plasma in the presence and absence of anti-TPT antibodies. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Nova-Pak C18 column (3.9 mm × 150 mm, 4 μm) protected by a Nova-Pak C18 guard column (3.9 mm × 20 mm, 4 μm), where 10 mM KH 2PO 4–methanol–triethylamine (72:26:2 (v/v/v), pH 3.5) was used as the mobile phase. Topotecan was quantified with fluorescence detection using an excitation wavelength of 361 nm and an emission wavelength of 527 nm. The retention time for the internal standard, acridine, and TPT were 7.4 and 9.0 min, respectively. The lower limit of quantitation (LOQ) for TPT was determined as 0.02 ng in mouse plasma and mouse plasma ultrafiltrate, corresponding to a concentration of 1 ng/ml in 20 μl mouse plasma. The assay was shown to be linear over a concentration range of 1–500 ng/ml. The recoveries of free and total TPT from spiked mouse plasma were within 10% of theoretical values (assessed at 1, 20 and 500 ng/ml). The validated HPLC assay was applied to evaluate TPT pharmacokinetics following administration of TPT to Swiss Webster mice and to hyperimmunized and control BALB/c mice. The assay has been shown to be capable for measuring total and free TPT in mouse plasma with high sensitivity and will allow the testing of the effect of anti-TPT antibodies on the disposition of TPT.

Determination of linezolid in plasma and bronchoalveolar lavage by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method

The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic assay for the determination of linezolid in human plasma, and bronchoalveolar lavage. The sample extraction was based on a fully automated solid-phase extraction with an OASIS HLB cartridge. The method used ultraviolet detection set at a wavelength of 254 nm and a separation with a Zorbax Eclipse XDB C8 column. The assay has been found linear over the concentration range 0.02–30 μg/ml and 0.04–30 μg/ml for linezolid, respectively, in plasma and bronchoalveolar lavage. It provided good validation data for accuracy and precision (CV <4.64% and 5.08%, accuracy in the range 96.93–102.67% and 97.33–105.67%, respectively, for intra- and inter-day). The assay will be applied to determine the penetration of linezolid in human bronchoalveolar lavage during pharmacokinetic steady-state.

Simultaneous quantitative analysis of ketanserin and ketanserinol in plasma by RP-HPLC with fluorescence detection.

A sensitive and selective high-performance liquid chromatographic assay for the quantification of ketanserin and ketanserinol in human plasma was developed and validated. The procedure involves extraction of ketanserin and ketanserinol from plasma using an Extrelut NT-1 solid-phase extraction column. The chromatograph was equipped with a Hypersil BDS column (100 x 4.5 mm, 3 micro m particle size). Separation was performed with a mixture of acetate buffer 0.01 M, pH 4.9-methanol-acetonitrile (52:40:8, v/v/v). Detection was performed with fluorescence detection (lambda(ex) = 332 nm and lambda(em) = 410 nm). Calibration curves were linear (r(2) = 0.999) in the range 0-400 ng/mL for both ketanserin and ketanserinol. The repeatability coefficient for ketanserin and ketanserinol was 3.1 and 3.0%, respectively. The reproducibility coefficient for ketanserin and ketanserinol was 10.5 and 9.1%, respectively. The limit of quantification for both ketanserin and ketanserinol was 2.0 ng/mL. The mean recovery yield for both ketanserin and ketanserinol was 60%. In an 8 h work day approximately 60 samples, including calibration and reference standards, could be processed.

Determination of levofloxacin in plasma, bronchoalveolar lavage and bone tissues by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method

The aim of this study was to develop a specific and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of levofloxacin in human plasma, bronchoalveolar lavage and bone tissues. The sample extraction was based on a fully automated liquid–solid extraction with an OASIS cartridge. The method used ultraviolet detection set at a wavelength of 299 nm and a separation with a Supelcosil ABZ+ column. The assay has been found linear over the concentration range 0.25–25 μg/ml for levofloxacin in plasma, 1–6 μg/ml in bronchoalveolar lavage and 0.5–10 μg/g for bone tissues and it provided good validation data for accuracy and precision. The assay will be applied to determine the penetration of levofloxacin in human bronchoalveolar lavage (BAL) and bone tissues during pharmacokinetic steady state.

Development of a bioanalytical liquid chromatography method for quantitation of 9-nitrocamptothecin in human plasma

9-Nitrocamptothecin (9-NC) is an orally administered camptothecin (CPT) that is under evaluation in clinical trials. This compound is not fluorescent, which has hampered development of a sensitive high-performance liquid chromatographic (LC) assay for measurement of drug concentrations in clinical trials. We now report development of an assay that involves reduction of 9-NC to the fluorescent compound 9-aminocamptothecin (9-AC). The method is based on enzymatic reduction of 9-NC using bovine liver S-9 fraction. This method is validated to quantitate 9-NC and 9-AC in patient samples, and yields results comparable to those obtained with an LC/MS method.

Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation

There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5′-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories ( N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects ( N=126) were 56 (21–138) nmol/l and 410 (250–680) pmol/g Hb, respectively. There was a strong positive correlation ( r 2=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.

Sensitive assay for the determination of fluvastatin in plasma utilizing high-performance liquid chromatography with fluorescence detection.

The authors describe a rapid, useful, specific, and very sensitive high-performance liquid chromatographic assay for the determination of fluvastatin (FV) level using atorvastatin as the internal standard (IS). After a simple deproteinization of 1.0 mL of plasma with acetonitrile, the drug and IS were extracted with tert-methyl butyl ether (TMBE). An efficient separation was performed using an 8 mm x 10 cm Nova Pak C(18) 4-microm particle size radial compression cartridge. The mobile phase consisted of an aqueous solution containing 20 mmol/L dibasic sodium dihydrogen phosphate with 1 mmol/L sodium lauryl sulfate adjusted to pH 7 with phosphoric acid and acetonitrile (70:30 v/v) delivered at a flow rate of 1.0 mL/min. The compounds of interest were detected using a fluorescence detector with the excitation wavelength set at 305 nm and the emission at 380 nm. Under these conditions, the retention times for FV and IS were 8.8 and 10.6 minutes, respectively. The concentration of FV in plasma was linear (r > 0.999) for the wide range that was examined (0.5-1,000 ng/mL). The recovery ranged from 88% to 96%. This sensitive, rapid, and simple analytical method gives accurate results over the wide range of concentrations examined. This method is used currently for clinical therapeutic monitoring and pharmacokinetic studies of FV in patients with hypercholesterolemia.

Quantitation of paclitaxel in micro-sample rat plasma by a sensitive reversed-phase HPLC assay

A sensitive high-performance liquid chromatographic (HPLC) method was developed for the determination of paclitaxel in micro-samples of rat plasma in order to study the mechanism of enhanced systemic exposure of paclitaxel co-administered with P-glycoprotein inhibitors. The assay involved solid-phase extraction procedures using 2′-methylpaclitaxel as the internal standard. Chromatographic separations were achieved using a ZORBAX ODS C18 column and mobile phase consisting of acetonitrile, methanol and ammonium acetate buffer (10 mM, pH 5.0) (48.5:16.5:35) pumped at 0.8 ml/min. The effluents were measured for UV absorption at 227 nm, with retention times of 8.5 and 11.0 min for paclitaxel and 2′-methylpaclitaxel, respectively. The chromatographic separation was excellent, with no endogenous interference. The standard curves showed a good linearity ( r=0.9994) over the concentration ranges of 10–1000 ng/ml. At 1000 ng/ml, the absolute recoveries of paclitaxel and 2′-methylpaclitaxel are 89 and 90%, respectively. The intra- and inter-day variabilities of paclitaxel were both less than 15%. This validated method for the assay of paclitaxel in micro-sample rat plasma made it feasible to study the pharmacokinetics of the drug in a single rat.

Determination of α-naphthylisothiocyanate and metabolites α-naphthylamine and α-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of α-naphthylisothiocyanate (1-NITC) and two metabolites α-naphthylamine (1-NA) and α-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C 18 5-μm column, a mobile phase of acetonitrile–water (ACN–H 2O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95–106 and 97–103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at −20 and −80 °C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 °C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague–Dawley rat.