Simultaneous determination of GTS-21 and its metabolite in rat plasma by high-performance liquid chromatography using solid-phase extraction

It has been suggested that GTS-21 can improve the learning deficits and inhibit the neuro-degeneration in patients with Alzheimer's disease. This paper describes a reversed-phase high-performance liquid chromatographic assay with visible detection at 405 nm for determination of GTS-21 and its metabolite, 4-hydroxy-GTS-21 in rat plasma. The method uses solid-phase extraction with a Bond Elut C₁₈ column. A quantitation limit of 1.0 ng/ml was achieved using 0.5 ml of rat plasma. In the validation study, the coefficients of variation and the relative errors of each compound were less than 10%. Also freeze-thaw and storage stability were confirmed. This method has proved to be applicable to the pharmacokinetic study of GTS-21 in rats.     

Assay for the quinocarmycin analog DX-52-1 in human plasma using high-performance liquid chromatography with automated column switching and low wavelength ultraviolet detection

The hydrocyanated derivative of the antitumor antibiotic quinocarmycin, DX-52-1 (I), exhibits impressive activity against human melanoma xenograft models in vivo. Phase I clinical trials to evaluate this compound as an antineoplastic agent have been initiated by the US National Cancer Institute. We have developed an HPLC assay for the determination of I in human plasma involving automated column switching and UV detection at 210 nm. The preparation of samples for chromatographic analysis entails the preliminary removal of plasma proteins by precipitation with acetonitrile, acidifying the clear supernatant to pH 4.5, then extracting twice with tert.-butyl methyl ether to recover the drug. A heart-cutting procedure employing two HPLC columns with contrasting retention characteristics under isocratic reversed-phase conditions was used to achieve the selectivity required for low wavelength UV detection of the analyte. The sample extract was initially loaded onto a column packed with a cyanopropyl stationary phase. During the predetermined time interval that I eluted from this column, a fully automated six-position switching valve was used to direct the effluent onto an octadecylsilane analytical column. The assay has been thoroughly validated with regard to linearity, inter- and intra-day accuracy and precision, recovery, selectivity and specificity. Using a sample volume of 1.0 ml, the lowest concentration of I quantified with acceptable day-to-day reproducibility was found to be 2.56 ng/ml (R.S.D. 18.9%, n=21, 4 months). This proved to be sufficiently sensitive for pharmacokinetic drug level monitoring in cancer patients treated with a 6-h continuous intravenous infusion of I, even at the starting dose of 3 mg/m². The successful performance and reliability of the assay has been demonstrated through extensive application to the routine analysis of plasma specimens acquired during a phase I clinical trial of the drug.     

Determination of retinol and retinyl esters in human plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A HPLC method with automated column switching and UV detection is described for the simultaneous determination of retinol and major retinyl esters (retinyl palmitate, retinyl stearate, retinyl oleate and retinyl linoleate) in human plasma. Plasma (0.2 ml) was deproteinized by adding ethanol (1.5 ml) containing the internal standard retinyl propionate. Following centrifugation the supernatant was directly injected onto the pre-column packed with LiChrospher 100 RP-18 using 1.2% ammonium acetate–acetic acid–ethanol (80:1:20, v/v) as mobile phase. The elution strength of the ethanol containing sample solution was reduced by on-line supply of 1% ammonium acetate–acetic acid–ethanol (100:2:4, v/v). The retained retinol and retinyl esters were then transferred to the analytical column (Superspher 100 RP-18, endcapped) in the backflush mode and chromatographed under isocratic conditions using acetonitrile–methanol–ethanol–2-propanol (1:1:1:1, v/v) as mobile phase. Compounds of interest were detected at 325 nm. The method was linear in the range 2.5–2000 ng/ml with a limit of quantification for retinol and retinyl esters of 2.5 ng/ml. Mean recoveries from plasma were 93.4–96.5% for retinol (range 100–1000 ng/ml) and 92.7–96.0% for retinyl palmitate (range 5–1000 ng/ml). Inter-assay precision was ≤5.1% and ≤6.3% for retinol and retinyl palmitate, respectively. The method was successfully applied to more than 2000 human plasma samples from clinical studies. Endogenous levels of retinol and retinyl esters determined in female volunteers were in good accordance with published data.     

Development of a high-performance liquid chromatographic–mass spectrometric assay for the specific and sensitive quantification of Ro 64-0802, an anti-influenza drug, and its pro-drug, oseltamivir, in human and animal plasma and urine

Oseltamivir phosphate (Ro 64-0796/002) is a pro-drug of the anti-influenza neuraminidase inhibitor, Ro 64-0802, and as Tamiflu™, has been developed for the treatment of both A and B strains of the disease. This paper describes an HPLC–MS–MS assay for both compounds in plasma and urine which fulfils all of the criteria for a good analytical method. It is sensitive with limits of quantification of 1 and 10 ng/ml for the pro-drug and active neuraminidase inhibitor, respectively. It is both accurate and precise with typical coefficients of variation from some 5000 quality control samples of approximately ±3 and ±6%, respectively. Extensive stability studies have demonstrated the absence of significant problems associated with the decomposition of either compound, although ex vivo hydrolysis of Ro 64-0796 to Ro 64-0802 in rodent plasma has to be prevented by the use of the esterase inhibitor, dichlorvos.     

Automated quantitative determination of a new polyamine biosynthesis inhibitor (CGP 48 664) and a potential metabolite in human and animal plasma by high-performance liquid chromatography

A specific and sensitive liquid chromatographic assay for the determination of 4-amidino-1-indanone-2′-amidinohydrazone (CGP 48 664, I) and a potential metabolite, 2-(4-carbamoyl-2,3-dihydro-1H-inden-1-yliden) hydrazine carboximidamide (CGP 53 391, II), in human and animal plasma was developed. CGP 51 467, a structural analog, was added to the plasma samples (up to 1 ml) as an internal standard. After mixing, the samples were processed automatically using an ASPEC solid-phase extraction system. The final extracts were chromatographed on a 5 μm Purospher RP-18 HPLC column. Chromatography was performed using a gradient system and UV detection. The described HPLC method is suitable for specific and quantitative measurement of concentrations of I, as well as its potential metabolite II down to 5–10 ng/ml in human and animal (dog, rat) plasma with acceptable reproducibility and accuracy.     

Quantitative determination of the angiotensin-converting enzyme inhibitor cilazapril and its active metabolite cilazaprilat in pharmaceuticals and urine by high-performance liquid chromatography with amperometric detection

A rapid and simple high-performance liquid chromatographic method with amperometric detection has been developed for the quantitation of cilazapril and its active metabolite and degradation product cilazaprilat in urine and tablets. The chromatographic system consisted of a μBondapak C₁₈ column, using a mixture of methanol–5 mM phosphoric acid (50:50, v/v) as mobile phase, which was pumped at a flow-rate of 1.0 ml/min. The column was kept at a constant temperature of (40±0.2)°C. Detection was performed using a glassy carbon electrode at a potential of 1350 mV. Sample preparation for urine consisted of a solid-phase extraction using C₈ cartridges. This procedure allowed recoveries greater than 85% for both compounds. The method proved to be accurate, precise and sensitive enough to be applied to pharmacokinetic studies and it has been applied to urine samples obtained from four hypertensive patients (detection limit of 50 ng/ml for cilazapril and 40 ng/ml for cilazaprilat in urine). Results were in good agreement with pharmacokinetic data.     

Determination of nelfinavir, a potent HIV protease inhibitor, and its active metabolite M8 in human plasma by high-performance liquid chromatography with photodiode-array detection

We developed and characterized a high-performance liquid chromatographic assay for the determination of nelfinavir (NFV), a potent HIV protease inhibitor, and its active metabolite M8 in human plasma. Extraction of the internal standard, M8 and NFV from the plasma buffered at pH 9.5 was achieved by a liquid–liquid extraction with a mixture of methyl-tert.-butyl ether and hexane. Following two washes of the reconstituted sample with hexane, separation was achieved on an octadecylsilyl analytical column with a mobile phase containing 0.1% trifluoroacetic acid–acetonitrile–methanol (51:46:5, v/v). Detection was performed using an ultraviolet photodiode-array detector. The signal was monitored at a wavelength of 220 nm. The assay was found to be linear and has been validated over the concentration range of 25 to 3000 μg/l for M8 and 25 to 6000 μg/l for NFV, from 500 μl of plasma. Recoveries were 98.9% (SD 8.9%), and 100.2% (SD 11.7%) for M8 and NFV, respectively. Concentrations that gave a signal-to-noise ratio of three (15 μg/l for both M8 and NFV) were selected to determine the limit of detection. The lower limit of quantification (25 μg/l for both M8 and NFV) was defined as the concentration for which the relative standard deviation and the percent deviation from the nominal concentration were lower than 20%.     

Determination of a new podophyllotoxin derivative, TOP-53, and its metabolite in rat plasma and urine by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method was developed for the determination of a new podophyllotoxin derivative, TOP-53 (I), and TOP-53 glucoronide (II) as its major metabolite in rat plasma and urine. For the analysis of I, the sample was chromatographed on a reversed-phase C₁₈ column with electrochemical detection after consecutive two-step liquid-liquid extractions. Compound II was determined as I after enzymatic hydrolysis of II. This method was validated sufficiently with respect to specificity, accuracy, and precision. The limiits of quantitation for both I and II were 2 ng/ml in plasma and 10 ng/ml in urine. The method is thus useful for the pharmacokinetic study of I.     

Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma is described. TPI was isolated from biological samples by solid-phase extraction on Bond Elut PRS columns. Chromatographic separation was achieved on a C₁₈ column using a mobile phase consisting of acetonitrile–10 mM acetate buffer (pH 4.3) including hexanesulfonate, with UV detection at 276 nm. This method has been validated across the range of 50–50 000 ng/ml using a 0.1-ml plasma volume. The mean recoveries from spiked plasma were 93% for dog and 94% for rat, respectively. The accuracy, precision and specificity of the method were demonstrated to be acceptable, and it was applied to the toxicokinetic study of TPI in rats.     

Determination of a peptide-doxorubicin,prostate-specific antigen activated prodrug,and its active metabolites in human plasma using high-performance liquid chromatography with fluorescence detection. Stabilization of the peptide prodrug with EDTA

A method for the determination of I, a peptide-doxorubicin conjugate that was evaluated for the treatment of prostate cancer, and two of its active metabolites, doxorubicin and leucine-doxorubicin is described. Blood samples were chilled immediately after being drawn in order to prevent ex vivo entry of the metabolites into red blood cells. EDTA (10 mg/ml final concentration) was used to prevent plasma-mediated degradation of the peptide portion of the prodrug. After the addition of internal standard, plasma was prepared for analysis using a C-8 solid-phase extraction column. In order to overcome secondary ionic interactions with the silica-based extraction column, the analytes were eluted with ammonium hydroxide in methanol. The extracts were evaporated to dryness, reconstituted, and assayed by step change, gradient, reverse phase HPLC with fluorescence detection. Two interfering metabolites found in post dose plasma were chromatographically separated by an adjustment of the mobile phase pH. The within-day reproducibility of the doxorubicin and leucine-doxorubicin chromatographic retention times was improved by a brief washing of the analytical column with 90% acetonitrile after each injection. The range of the standard curve was 12.5-1250 ng/ml for doxorubicin and 25-2500 ng/ml for I and leucine-doxorubicin.     

Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography: Application to a pharmacokinetic study

This paper describes a relatively simple and sensitive high-performance liquid chromatographic assay (HPLC) with ultraviolet absorbance detection for 5-fluorouracil (5-FUra) and its two main metabolites, 5-fluorouridine (5-FUrd) and 5-fluoro-2′-deoxyuridine (5-FdUrd), in plasma. In this study, two plasma clean-up procedures involving addition of internal standard, solid-phase and liquid-liquid extractions have been developed. A reversed-phase Kromasil C₁₈ column was used. The detection was performed at 268 nm for 5-FUra and at 275 nm for the two metabolites. Linear detection responses were obtained for concentrations ranging from 25 to 1000 ng/ml. The average recovery from plasma was 35, 42 and 48% for 5-FUra, 5-FUrd and 5-FdUrd, respectively. Precision, expressed as C.V., ranged from 2.7 to 13% and the mean recovery from 94 to 105%. The limits of quantitation and detection of the three analytes were 20 and 10 ng/ml, respectively. The method was used to monitor the pharmacokinetic profile of 5-FUra and its two metabolites in patients with metastatic colorectal cancer.     

Determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and ultraviolet detection

A highly sensitive HPLC method with automated column switching was developed for the simultaneous determination of endogenous levels of 13-cis-retinoic acid (isotretinoin), all-trans-retinoic acid (tretinoin) and their 4-oxo metabolites in plasma samples from man, Cynomolgus monkey, rabbit, rat and mouse. Plasma (0.4 ml) was deproteinated by adding ethanol (1.5 ml) containing the internal standard acitretin. After centrifugation, 1.4 ml of the supernatant were directly injected onto the precolumn packed with LiChrospher 100 RP-18 (5 μm). 1.25% ammonium acetate and acetic acid-ethanol (8:2, v/v) was used as mobile phase during injection and 1% ammonium acetate and 2% acetic acid-ethanol (102:4, v/v) was added, on-line, to decrease the elution strength of the injection solution. After backflush purging of the precolumn, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm. Two coupled Superspher 100 RP-18 endcapped columns (both 250×4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid: (A) 600:300:60:10 (v/v/v/v), (B) 950:20:5:20 (v/v/v/v), and (C) 990:5:0:5 (v/v/v/v). The method was linear in the range 0.3–100 ng/ml, at least, with a quantification limit of 0.3 ng/ml. The mean recoveries from human plasma were 93.2%–94.4% and the mean inter-assay precision was 2.8%–3.2% (range 0.3–100 ng/ml). Similar results were obtained for animal plasma. The analytes were found to be stable in the plasma of all investigated species stored at −20°C for 4.3 months and at −80°C for 9 months, at least. At this temperature, human plasma samples were even stable for 2 years. The method was successfully applied to more than 6000 human and 1000 animal plasma samples from clinical and toxicokinetic studies. Endogenous levels determined in control patients and pregnant women were similar to published data from volunteers.