Reversed-phase High-performance Liquid Chromatographic Assay Research Articles

Determination of topotecan in human and mouse plasma and in mouse tissue homogenates by reversed‐phase high‐performance liquid chromatography

A sensitive and selective reversed-phase high-performance liquid chromatographic (HPLC) assay has been developed and validated for quantification of total topotecan in human and mouse plasma and in mouse tissue samples. Isocratic separation was achieved on a Zorbax SB-C(18) column and topotecan was monitored fluorimetrically. Two ranges of calibrations curves were used to determine lower levels of topotecan more accurately. Acceptable accuracy and precision was achieved for all matrices. Topotecan was stable upon repeated freeze-thawing for three cycles or storage for 24 h at ambient temperatures in spiked plasma samples and tissue homogenates, except in heart homogenates. In an additional validation experiment in which (14)C-labeled topotecan was administered to mice, the levels of unchanged topotecan were about 80-90% of the total radioactivity in tissue homogenates collected 10 min after drug administration and virtually similar as in plasma samples. However, results in tissue homogenates obtained 4 h post-drug administration indicated substantial metabolism of topotecan. This assay is suitable for studying the pharmacokinetics and tissue distribution of topotecan in mice. Our results demonstrate the importance of including all tissues of interest for pharmacokinetic studies in the validation procedure.

Determination of calycosin‐7‐O‐β‐d‐glucopyranoside in rat plasma and urine by HPLC

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.

Coadministration of oxaliplatin does not influence the pharmacokinetics of gemcitabine

We investigated the possible pharmacokinetic interactions of gemcitabine and oxaliplatin in patients with advanced solid tumors. Ten patients with advanced stage solid tumors were treated with gemcitabine (1500 mg/m) as a 30-min intravenous infusion on days 1 and 8, followed by oxaliplatin (130 mg/m) as a 4-h intravenous infusion, on day 8 every 21 days. Pharmacokinetic data for 24 h after dosing were obtained for both day 1 (gemcitabine without oxaliplatin coadministration) and day 8 (gemcitabine with oxaliplatin) during the first cycle of treatment. Gemcitabine levels in plasma were quantified using a reverse-phase high-performance liquid chromatography assay with ultraviolet detection, and total and ultrafiltrated platinum levels by flameless atomic absorption spectrophotometry with deuterium correction. All pharmacokinetic parameters of gemcitabine seemed to be unchanged when coadministered with oxaliplatin (day 8) compared with pharmacokinetic data of gemcitabine given as a single agent (day 1). The mean (maximum) concentration of gemcitabine on days 1 and 8 was 13.57 (+/-7.42) and 10.23 (+/-5.21) mg/l, respectively (P=0.28), and the mean half-life was 0.32 and 0.44 h, respectively (P=0.40). Similarly, the P-values for AUC0-24 and the observed clearance were 0.61 and 0.30, respectively. Plasma total and free platinum levels were in agreement with other published data. Gemcitabine disposition appeared to be unaffected by oxaliplatin coadministration because no significant changes in pharmacokinetics between day 1 (gemcitabine without oxaliplatin coadministration) and day 8 (gemcitabine with oxaliplatin) were observed.

Haemagglutinin quantification and identification of influenza A&B strains propagated in PER.C6 ® cells: A novel RP-HPLC method

The major antigenic determinant of influenza A and B virus is haemagglutinin (HA). The HA content is an important specification of influenza vaccines. HA in vaccines has typically been quantified by single-radial-immunodiffusion (SRID). However, SRID is a laborious and low throughput assay. Moreover, sensitivity, accuracy, and precision, especially for non-purified (in-process) influenza virus is relatively low. We present a novel method for quantification of HA in influenza viral cultures as well as for the identification of HA from individual influenza strains in trivalent vaccines. The method is based on the separation of HA 1, the hydrophilic subunit of HA, from the more hydrophobic viral and matrix components by reversed-phase high performance liquid chromatography (RP-HPLC). The HA 1 peak area is demonstrated to be proportional to the level of HA in non-purified, semi-purified and purified vaccine products of various epidemic and pandemic influenza A and B strains propagated in PER.C6 ® cell cultures. The RP-HPLC assay selectivity allows for the simultaneous identification and quantification of HA 1 from influenza A and B strains in the yearly revised trivalent vaccines for epidemic outbreaks.

Determination of Amprenavir Total and Unbound Concentrations in Plasma by High-Performance Liquid Chromatography and Ultrafiltration

Amprenavir is an HIV-1 protease inhibitor with high protein binding (90%) in human plasma. This study was designed to develop an assay to measure the concentration of unbound amprenavir, to study variation with time in patients, and to investigate whether ritonavir and lopinavir, other protease inhibitors that could be combined, interact with amprenavir protein binding in vitro. A reverse-phase high-performance liquid chromatography assay to UV detection was developed and validated to measure total amprenavir in plasma, and this method was adapted to quantitate low concentrations of unbound amprenavir in ultrafiltrate aqueous fluid. Equilibrium dialysis and ultrafiltration were used and compared with separate unbound fraction. The latter method was easier to use and was, therefore, subsequently adopted. In 10 patients who received amprenavir 600 mg bid combined with ritonavir, mean amprenavir free-fraction in plasma was 8.6% (range, 4.4-20%). When added to pooled human plasmas at concentrations close to those found in treated patients, the unbound amprenavir fraction was increased in the presence of lopinavir, but remained unaffected by ritonavir. It remains to be seen whether measurement of unbound concentrations, rather than total concentrations, could improve therapeutic drug monitoring.

High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urine

A reversed-phase high-performance liquid chromatography assay for mangiferin in rat plasma and urine was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) containing 3% acetic acid at a flow rate of 1 mL/min. Detection was at 257 and 365 nm for mangiferin in plasma and urine, respectively. The limit of quantitation (LOQ) of mangiferin was 0.6 microg/mL in plasma, and 0.48 microg/mL in urine. The standard curve was linear from 0.6 to 24 microg/mL in plasma, and 0.48 to 24 microg/mL in urine, both intra- and inter-day precision of the mangiferin were determined and their RSD did not exceed 10%. The method provides a technique for rapid analysis of mangiferin in rat plasma and urine, which can be used in pharmacokinetic studies.

Penetration of Orally Administered Prulifloxacin Into Human Lung Tissue

To evaluate the distribution in lung tissue of ulifloxacin, the active metabolite of prulifloxacin, a new once-daily fluoroquinolone administered orally in a single 600mg dose. Open-label, randomised study. Twenty-seven patients (25 males, 2 females; mean age 65.7 years [range 49-79 years]) with a lung neoplasm requiring lobectomy or pneumonectomy. Patients were randomly assigned to five treatment groups and received a single oral dose of prulifloxacin 600mg at 2, 4, 6, 12 or 24 hours preoperatively. During surgery, blood and healthy lung (based on macroscopic appearance) samples were collected at the same time. Ulifloxacin concentrations in plasma and lung tissue were determined by a validated reversed-phase high-performance liquid chromatography assay. Lung tissue ulifloxacin concentrations were adjusted for blood contamination, by measuring haemoglobin in the supernatant of each tissue sample and applying a corrective equation. Ulifloxacin concentration in lung tissue exceeded plasma concentration at every timepoint. Following administration of prulifloxacin 600mg, the overall mean corrected lung/plasma ratio over the 24-hour period was 6.9 (range 1.2-14.1). When sampling intervals were assessed, the corrected lung/plasma ratios were 7.5 (2 hours after dosing), 6.3 (4 hours), 4.3 (6 hours), 7.0 (12 hours) and 9.2 (24 hours). The mean corrected lung/plasma area under the concentration-time curve ratio was 6.3, demonstrating the ability of the drug to penetrate lung tissue and confirming the high exposure of this target tissue to ulifloxacin. However, the limitation of the lung tissue sampling method and the high interpatient variability should be considered. Over the 24-hour period, the concentrations of ulifloxacin in lung tissue were higher than the minimum inhibitory concentration (MIC) values for pathogens frequently involved in community-acquired respiratory tract infections. Lung tissue penetration data may have a supportive value when considered jointly with MICs and efficacy results. The findings from this lung penetration study could explain the efficacy of once-daily prulifloxacin 600mg observed in clinical trials conducted in patients with exacerbation of chronic bronchitis.

A chromatographic method for baicalin quantification in rat thalamus

A rapid reversed-phase high-performance liquid chromatographic (rp-HPLC) assay for the determination of baicalin in rat thalamus was developed. This was carried out on a Hypersil -C(18) column using 4-nitro-benzoic acid as the internal standard with a mobile phase of methanol-water-H(3)PO(4) (45:55:0.2, v/v/v). Detection was by UV at 277 nm. The calibration curve for baicalin was linear (r=0.9992) over the concentration range of 0.05--4.0 microg/mL and the limit of detection was 10 ng/mL. The coefficients of variation of intra- and inter-day assays were 2.64, 5.19 and 3.19% and 3.46, 6.21 and 5.58% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The recoveries of baicalin from rat thalamus were 85.4+/- 5.62, 90.7+/- 2.43 and 89.1+/- 4.75% at concentrations of 0.5, 1.0 and 3.0 microg/mL, respectively. The method was applied to determine the time course of baicalin in rat thalamus, following a single dosage of intravenous administration of Scutellariae radix extract at 90 mg/kg of baicalin to male Wistar rats.

A simple high-performance liquid chromatographic method for detection of hydroxyzine in human plasma after overdose

Introduction: Hydroxyzine, a piperazine H1-receptor antagonist, is effective in generalised anxiety disorder. For toxicological purposes, a simple reversed-phase high-performance liquid chromatographic assay was developed for the detection of hydroxyzine in human plasma. Methods: A liquid–liquid procedure was used to extract the drug from plasma in the presence of an internal standard (clothiapine). The analysis was performed on a Spherisorb S5 C 8 analytical column with UV detection. Results: A linear response was observed over the concentration range 20–1500 ng/ml. A good accuracy (bias<7.3%) was achieved for all quality controls, with intraday and interday variation coefficients inferior to 8.5%. The limit of detection was 10 ng/ml, without interference of endogenous components. Discussion: This rapid method (run time<13 min) is currently used for poison management involving hydroxyzine.

Stabilization and HPLC Analysis of Betamethasone Sodium Phosphate in Plasma

The analysis of corticosteroid prodrugs in pharmacokinetic (PK) studies poses the risk of overestimation of corticosteroid concentrations due to in vitro hydrolysis of prodrugs after sample collection. This study tests the effectiveness of enzyme inhibitors as stabilizers for betamethasone sodium phosphate (BSP) in pregnant sheep plasma samples collected during PK studies with betamethasone (BET) and provides simultaneous high-performance liquid chromatography analysis of BSP and BET. A rapid, sensitive, and specific ion-paired reversed-phase high-performance liquid chromatography assay for simultaneous measurement of BET and BSP in plasma was developed. This assay was used for analyzing samples from an in vitro prodrug hydrolysis study. Enzyme inhibitors tested were sodium arsenate (Na2HAsO4) and ethylenediaminetetraacetic acid. The BSP was administered intramuscularly to three pregnant sheep to assess in vivo PK. Samples were split with part treated with Na2HAsO4 and part left natural. In vitro hydrolysis of BSP in plasma to BET could be completely inhibited by Na2HAsO4, but not by ethylenediaminetetraacetic acid. The PK study showed lower concentrations of BET in samples with Na2HAsO4 compared with natural samples. This study demonstrates that artifacts in PK profiles of corticosteroids due to in vitro prodrug hydrolysis can be prevented by sample treatment with enzyme inhibitors. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 μg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (≥150 μl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (<0.05 μg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025–10 μg/ml) and quality control samples (0.1, 0.5, and 2.5 μg/ml) were <6.2% coefficient of variation and 96.8–104.0% of nominal concentration, respectively.

Simultaneous determination of six HIV nucleoside analogue reverse transcriptase inhibitors and nevirapine by liquid chromatography with ultraviolet absorbance detection

An accurate, sensitive and specific reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of the nucleoside reverse transcriptase inhibitors zalcitabine, lamivudine, didanosine, stavudine, zidovudine, and abacavir with the non-nucleoside reverse transcriptase inhibitor nevirapine in human blood plasma is described. The new Polarity dC C 18 silica column used in this method provides better resolution and peak shape than all other columns tested. Also, four different ultraviolet wavelengths were used for accurate and specific quantitation of the analytes. The method was validated over the range of 10–10 000 ng/ml for all analytes except zalcitabine (10–5000 ng/ml). This method is accurate (average accuracies of three different concentrations ranged from 97.2 to 105%), and precise (within- and between-day precision measures ranged from 0.5 to 5.1% and 0.5 to 5.6%, respectively), and is currently being used for determination of plasma drug concentrations in our laboratory.

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.

Reversed-phase high performance liquid chromatography of the products of microbiological degradation of toluene

Conditions have been selected for a reversed-phase high-performance liquid chromatographic assay of intermediate products formed in the course of utilization of toluene by Pseudomonas putida. The composition of products indicates that degradation of toluene by strain BS590-P proceeds primarily through the formation of benzoate and catechol. This is followed by degradation of catechol via ortho-cleavage. In strain BS3701-P, toluene oxidation involves both the side chain and the aromatic ring.

Simultaneous determination of oleuropein and hydroxytyrosol in rat plasma using liquid chromatography with fluorescence detection

Oleuropein, the main glycoside present in olives, and hydroxytyrosol, the principal degradation product of oleuropein present in olive oil, have been linked to reduction of coronary heart disease and certain cancers. In the present study a direct and sensitive reversed-phase high-performance liquid chromatographic assay was developed for simultaneous quantification of both oleuropein and hydroxytyrosol. The plasma protein was precipitated with acetonitrile, samples were then centrifuged and supernatants were dried, and reconstituted with water prior to injection. The chromatographic analysis was carried out using a phenyl column and an isocratic elution of acidified water and acetonitrile with fluorescence detection at 281 and 316 nm for excitation and emission, respectively. The calibration curve was linear and limits of quantification were 30 ng/ml and 3 μg/ml for hydroxytyrosol and oleuropein, respectively. The method has been successfully applied to monitor oleuropein and hydroxytyrosol plasma levels in the rat.

Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and automation

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE ® II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE ® II EX robotic liquid handling system.

Simple and rapid quantification of the non-nucleoside reverse transcriptase inhibitors nevirapine, delavirdine, and efavirenz in human blood plasma using high-performance liquid chromatography with ultraviolet absorbance detection

A simple reversed-phase high-performance liquid chromatography assay for the simultaneous quantitative determination of three HIV non-nucleoside reverse transcriptase inhibitors (nevirapine, delavirdine, and efavirenz) in human blood plasma is described. The method was validated over the range of 10 ng/ml to 50 μg/ml for nevirapine, 25 ng/ml to 25 μg/ml for delavirdine, and 10 ng/ml to 10 μg/ml for efavirenz. The method is accurate (average accuracies over eight concentrations ranging from 87.3 to 113%), and precise (within-day and between-day precision measures ranging from 0.12 to 7.9% and 0.26 to 5.9%, respectively). All three non-nucleoside reverse transcriptase inhibitors proved to be stable under various conditions. Due to its simplicity, this assay can readily be used for investigational or clinical monitoring of plasma concentrations.

Thiopurine methyltransferase activity: new conditions for reversed-phase high-performance liquid chromatographic assay without extraction and genotypic–phenotypic correlation

Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine drugs. A genetic polymorphism is responsible for large inter-individual differences observed in TPMT activity. We report a new HPLC technique, which avoids an extraction step and the use of radioactive reagents, based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl- l-methionine (SAM) as methyl donor in red blood cell lysates (RBC). Intra- and inter-assay variation, within-day, within-run, between-day, and between-run variations showed high precision. The formation of 6-MMP was linear with respect to the lysate concentration and time. In a blinded assay of 61 samples, the results of HPLC method correlated with those of the radiochemical method ( r 2=0.82, P<0.0001). Using a cut-off point of 8.5 nmol/h/ml packed RBC, positive predictive value of HPLC was 100% for heterozygous patients. Because of the absence of extraction step, this new HPLC technique of TPMT activity determination reduces analysis variation and is time-saving. This rapid, sensitive, and reproducible method is suitable for routine monitoring of TPMT activity and for fundamental studies.

Improved high performance liquid chromatographic determination of ginsenosides in Panax ginseng-based pharmaceuticals using a diol column.

A reversed-phase high-performance liquid chromatographic assay for the simultaneous quantitative determination of seven ginsenosides, Rb(1), Rb(2), Rc, Rd, Rg(1), Re and Rf in pharmaceutical preparations is described. Chromatographic separation was achieved in less than 20 min using a 250 x 4 mm Lichrospher, 5 microm, 100 A diol column with detection at 203 nm. The method was validated over the range of 2.5-20 ng/microL using a 20 microL sample volume. The average accuracy at five concentrations was 90-100%, and the within-day and between-day precision ranged from 1 to 7% expressed as coefficient of variation. The detection limit and the quantitation limit of the method were 20 and 50 ng injected for each ginsenoside, respectively.

Kinetics and mechanism of hydrolysis of efavirenz.

To determine the kinetics and mechanism of hydrolysis of efavirenz [(S)-6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-2H-3,1-benzoxazin-2-one] in aqueous solutions. The solution stability was examined at 60 degrees C and an ionic strength of 0.3 M over the pH range of 0.6 to 12.8. The loss of efavirenz and the appearance of degradants were followed with a reverse-phase high-performance liquid chromatography assay. Characterization of the degradants was accomplished with liquid chromatography-mass spectrometry. The degradation of efavirenz followed apparent first-order kinetics over the pH range of 0.6 to 12.8 at 60 degrees C. The catalytic effect of phosphate and borate buffers was negligible while acetate and citrate demonstrated buffer catalysis. The overall rate constant indicated a pH minimum (the pH of maximum stability) of approximately 4. Mass spectra data identified a degradant with a molecular weight consistent with hydrolysis of the cyclic carbamate to the corresponding amino alcohol. The degradation route was confirmed with spiking experiments with an authentic sample of the amino alcohol indicating that the carbamate hydrolysis pathway was the predominant reaction throughout the pH range studied. Subsequent degradation of the amino alcohol proceeded at the extremes of the pH range studied via rearrangement to the quinoline. The pH-rate profile was consistent with a combination of a V-shaped profile in the pH range of 0-9 and a sigmoid-shaped profile in the pH range of 4-13. The plateau that began at pH 10-11 is a result of the ionization of the amine of the carbamate inhibiting the base-catalyzed hydrolysis of efavirenz, given that the ionized form of the carbamate is resonance-stabilized toward hydroxide-catalyzed degradation. Thus, increasing the pH resulted in a parallel decrease in the unionized fraction and increase in hydroxide ion concentration resulting in a constant k(obs) value.