Determination of the antifungal agent, ketoconazole, in human plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic (HPLC) assay for the quantitative determination of ketoconazole, an orally active antifungal agent, in human plasma is described. After extraction of the drug from plasma, the compound is separated by HPLC using a reversed-phase column and detected by UV light at 205 nm. Quantitation is accomplished by external standardization and the determination of peak areas is performed with the aid of an integrating computer. The average recovery of ketoconazole over a concentration range of 0.1–20.0 μg/ml was 88.2 ± 4.07% S.D. The maximum sensitivity of the assay is less than 0.1 μg/ml. The assay is suitable for use in pharmacokinetic studies following the administration of therapeutic doses of ketoconazole to humans.     

Determination of a new antifungal agent, voriconazole, by multidimensional high-performance liquid chromatography with direct plasma injection onto a size-exclusion column

A fully automated method has been developed for the analysis of a new antifungal agent, voriconazole, in human plasma. Multidimensional chromatography was used with size-exclusion chromatography as the first step to separate plasma protein from the drug and internal standard which were then trapped on a precolumn of pellicular ODS. A reversed-phase column, Spherisorb ODS2, then separated drug and internal standard from one another and from remaining plasma components. With an injection of 0.56 ml plasma the limit of quantitation of the method was 5 ng/ml.     

Measurement of the anti-cancer agent gemcitabine in human plasma by high-performance liquid chromatography

A reversed-phase HPLC assay has been developed to determine the concentration of the anti-metabolite 2',2'-difluorodeoxycytidine (gemcitabine, dFdC) in human plasma over the concentration range of 0.5-150 microM (0.13-39.44 microg/ml), and 2',2'-difluorodeoxyuridine (dFdU), the deaminated, inactive metabolite, over the range of 1.0-227 microM (0.26-60 microg/ml). After the addition of 20 nmol 2'-fluorodeoxycytidine (FdC) as an internal standard, 0.5-ml samples of plasma were subjected to acetonitrile precipitation, followed by analysis using a gradient reversed-phase HPLC assay with UV detection. A Phenomenex Columbus C(18) column, 5 microm, 150 x 4.6 mm, and a Waters C(18), 4 microm, Nova-Pak Sentry guard column were used to achieve separation. FdC, dFdC and dFdU were monitored at 282, 269 and 258 nm, respectively, on a Waters 996 photodiode array detector. The mobile phase, run at a total flow-rate of 1.5 ml/min, was composed of two solvents: 50 mM ammonium acetate pH 5.0 in either 2% (solvent A) or 10% methanol (solvent B, v/v); 100% solvent A was run for 17 min, followed by a linear gradient to 100% solvent B over 14 min. FdC, dFdC and dFdU were resolved from endogenous compounds and had retention times of 13.6+/-0.5, 18.1+/-1.1 and 29.0+/-0.6 min, respectively. The assay was useful in measuring the plasma levels of both analytes in samples obtained from adult cancer patients participating in a Phase I trial of gemcitabine given as either a 1- or 2-h infusion weekly for 3 of 4 weeks.     

Determination of stavudine, a new antiretroviral agent, in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A sensitive high-performance liquid chromatographic assay has been developed to determine the levels of a new antiretroviral agent, stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T), in human plasma. Didanosine (2′,3′-dideoxyinosine, ddI) was used as the internal standard. The very selective sample pretreatment involved solid-phase extraction using silica gel columns. Chromatography was carried out on a μBondapak phenyl column, using a mobile phase of 0.005 M phosphate buffer (pH 6.8)—methanol (90:10, v/v) and ultraviolet detection at 265 nm. The method has been validated, and stability tests under various conditions have been performed. The detection limit is 10 ng/ml (using 500-μl human plasma samples). The bioanalytical assay has been used in a single pharmacokinetic experiment in a rat to investigate the applicability of the method in vivo.     

Measurement of resiniferatoxin in serum samples by high-performance liquid chromatography

A novel and simple method of extraction, separation, identification and quantification of resiniferatoxin (RTX) in serum samples is reported. Human serum and whole blood were treated with acetonitrile to denature proteins, such as orosomucoid, and the soluble fraction was passed through a reversed-phase C18 cartridge. RTX eluted from the cartridge was quantified by high-performance liquid chromatography (HPLC) using a reversed-phase C18 column. Reproducible recovery of RTX and tinyatoxin, an internal standard, from serum was achieved. Isocratic elution with 62% acetonitrile provided a suitable retention time without interfering peaks eluting near the analyte. Therefore, the procedure described provides a useful assay for determination of serum RTX pharmacokinetic parameters.     

Measurement of resiniferatoxin in cerebrospinal fluid by high-performance liquid chromatography

A sensitive and simple high-performance liquid chromatographic (HPLC) assay was developed for the quantification of resiniferatoxin (RTX) in canine cerebrospinal fluid (CSF). A reversed-phase C(18) column and acetonitrile in 0.02 M NaH(2)PO(4) as mobile phase provided satisfactory resolution for RTX analysis. Direct HPLC analysis of the CSF samples without sample extraction or preparation improves the accuracy and detection limits of this assay. This assay was applied to measure CSF RTX content to test this method for research and clinical applications related to studies examining its analgesia effects.     

Measurement of branched-chain ketoacids in plasma by high-performance liquid chromatography

Branched-chain ketoacids were isolated from plasma or serum samples by acidification, passage through a cationic exchange resin, ether extraction, and extraction of the ether layer with phosphate buffer. The recovery of 2-[1-14C]ketoisocaproate taken through these procedures averaged 95 +/- 3%. Branched-chain ketoacids were measured by high-performance liquid chromatography using a single mobile phase (sodium phosphate:acetonitrile). In normal human subjects, mean +/- SD fasting levels of 2-ketoisocaproate, 2-keto-3-methylvalerate, and 2-ketoisovalerate were 29 +/- 8, 18 +/- 4, and 12 +/- 3 microM, respectively. In normal rats, slightly different results were found: 24 +/- 10, 19 +/- 7 and 17 +/- 6 microM, respectively. In both species, levels of each ketoacid expressed as fractions of total branched-chain ketoacids were much less variable.     

Electrochemical detection for high-performance liquid chromatography of ketoconazole in plasma and saliva

Ketoconazole, cis-1-acetyl-4-[4[[2-(2,4-dichlorophenyl)-2-(1H-imidazol- 1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazine, a clinically used antifungal agent, is also an inhibitor of steroid hormone biosynthesis. A high-performance liquid chromatographic method is described which resolves ketoconazole with selectivity and high sensitivity provided by the use of electrochemical detection. Ketoconazole can be detected in high-performance liquid chromatography by electrochemical oxidation at a glassy carbon electrode at a potential of +1.0 V. Electrochemical detection offers improved sensitivity and selectivity over ultraviolet absorbance or fluorescence detection after derivatization. The method utilizes a volatile buffer system compatible with postcolumn analyses and an internal standard which is electrochemically active. This technique provides a simple method to assay ketoconazole. Ketoconazole can be detected in human plasma and saliva after a single oral therapeutic dose.     

Measurement of cyclomaltodextrin glucanotransferase activity by high-performance liquid chromatography using a fluorogenic substrate

A mixture of p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranoside (FG5P) and p-nitrophenyl alpha-D-glucoside (GP) was incubated with cyclomaltodextrin glucanotransferase (CGTase) [EC 2.4.1.19]. Analysis of the digest by HPLC showed that the products were p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D- glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-O-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P) and p-nitrophenyl alpha-D-maltoside (G2P), and no other product could be detected. Based on the reaction, a sensitive method to assay for CGTase was developed.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Determination of a new antibronchospastic agent, MX2/120, in guinea pig plasma by high-performance liquid chromatography in a pharmacokinetic study

7-[(2,2-Dimethyl)propyl)]-1-methylxanthine (I, Lab code MX2/120) is a new potent antibronchospastic agent. A rapid and simple HPLC assay for I in guinea pig plasma has been developed. Compound I was extracted from plasma with dichloromethane by a solid-phase extraction procedure, after adding 1,3-dimethyl-7-pentylxanthine at a concentration of 5 μg/ml as the internal standard (I.S.). The extraction residue was redissolved in water—acetonitrile and chromatographed on a RP-18 reversed-phase column. The eluate was monitored by spectrophotometric detection at 280 nm. The method showed good linearity over the range 0.1–20 μg/ml (r = 0.9998) and is precise (C.V. × Student's T-TEST = 1.84%) and accurate (mean recovery ± limit of CONFIDENCE = 100.25 ± 0.34). The HPLC assay was successfully applied to the determination of the pharmacokinetic profile of I after intravenous and oral administration in guinea pigs. The main pharmacokinetic parameters are presented.     

Measurement of genome wide DNA methylation by reversed-phase high-performance liquid chromatography

Intrinsic affinity tags are useful tools for the study of macromolecular targets. Although polypeptide affinity tags are routinely used in purification and detection of protein complexes, there has been a relative lack of powerful RNA affinity tags that can be embedded within RNA sequences. Here, the preparation and use of two RNA affinity tags against Sephadex or streptavidin are described. The two tags have different strengths that make them appropriate for slightly different uses. One is a high-affinity ligand for streptavidin that can be specifically eluted by competition with biotin under otherwise native binding conditions. The other tag binds selectively to Sephadex beads, and can be eluted by competition with the soluble dextran that composes Sephadex. When properly placed within another RNA molecule, the tags can be used to effect dramatic purification of RNA or ribonucleoprotein complexes from complex mixtures of cellular RNA.     

Highly sensitive analysis of the antifolate pemetrexed sodium, a new cancer agent, in human plasma and urine by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatography method was developed and validated for the quantitation of pemetrexed (LY231514, ALIMTA) in human urine and plasma. Plasma samples were spiked with the internal standard lometrexol and extracted using Certify II columns. Pemetrexed was assayed in diluted urine by an external calibration method. A C₈ column was used for the separation of analytes with a mobile phase composed of sodium formate buffer and acetonitrile. Between- and within-day precision and accuracy were acceptable down to the limit of quantitation of 5 ng/ml in plasma. This method was used successfully for an investigation of the disposition of pemetrexed in patients receiving 500 mg/m² as a 10-min infusion.     

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10³ concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole

The common skin disease acne vulgaris is caused by Propionibacterium acnes. A lipase secreted by this microorganism metabolizes sebum and the resulting metabolites evoke inflammation in human skin. The antifungal drug ketoconazole inhibits P. acnes lipase activity. We previously showed that the drug also inhibits the growth of P. acnes. Thus, ketoconazole may serve as an alternative treatment for acne vulgaris, which is important because the number of antibiotic‐resistant P. acnes strains has been increasing.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C₈ analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.