Determination of gestrinone in human serum by liquid chromatography–electrospray tandem mass spectrometry

A rapid, sensitive and specific high-performance liquid chromatography–electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5–177 ng/ml (r²≥0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3–13.7 to −4.8–3.0%. The inter-day precision and accuracy ranged from 5.5–14.8 to −6.7–3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.     

Determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in human urine

A method for simultaneous determination of 5-hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide in urine is described. These compounds are metabolites of N-methyl-2-pyrrolidone, a powerful and widely used organic solvent. 5-Hydroxy-N-methylpyrrolidone and 2-hydroxy-N-methylsuccinimide were purified from urine by adsorption to a C₈ solid-phase extraction column and then elution by ethyl acetate–methanol (80:20). After evaporation, the samples were derivatised at 100°C for 1 h by bis(trimethylsilyl)trifluoroacetamide. Ethyl acetate was then added and the samples were analysed by gas chromatography–mass spectrometry in the electron impact mode. The extraction recovery for 5-hydroxy-N-methylpyrrolidone was about 80% while that for 2-hydroxy-N-methylsuccinimide was about 30%. The intra-day precision for 5-hydroxy-N-methylpyrrolidone was 2–4% and the between-day precision 4–21% (4 and 60 μg/ml). The intra-day precision for 2-hydroxy-N-methylsuccinimide was 4–8% and the between-day precision 6–7% (2 and 20 μg/ml). The detection limit was 0.2 μg/ml urine for both compounds. The method is applicable for analysis of urine samples from workers exposed to N-methyl-2-pyrrolidone.     

Simultaneous determination of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma by reversed-phase high-performance liquid chromatography

A rapid, simple and sensitive high-performance liquid chromatographic (HPLC) assay has been developed for the simultaneous quantification of the HIV-protease inhibitors indinavir, amprenavir, ritonavir, saquinavir and nelfinavir in human plasma. The method involved the solid-phase extraction of the five drugs and the internal standard (I.S., verapamil) from 400 μl of human plasma. The HPLC analysis used a reversed-phase C₁₈ analytical column and a mobile phase consisting of a gradient with 15 mM phosphate buffer (pH 5.75)–acetonitrile and UV monitoring. The method was linear over the therapeutic concentration range for the five HIV-protease inhibitors. The accuracy of the method ranged from 98.2 to 106.7% and the precision values ranged from 1.4 to 8.1% for intra-day precision and from 3.1 to 6.4% for the inter-day values.     

Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

This paper describes the development of an isocratic reversed-phase high-performance liquid chromatographic method for the routine analysis of recombinant interleukin-2 (rIL-2) in liposome samples. The chromatographic system employed a C₄ column maintained at 30°C eluted with 52.5% (w/w) acetonitrile in water, containing 100 mM NaClO₄ and 10 mM HClO₄. To remove phospholipid interference the chromatographic method was combined with a lipid-extraction procedure. No significant loss of rIL-2 was noted upon inclusion of this extraction step. The protein eluted from the column with a capacity factor (k′) of 5.8. The method was validated for robustness, linearity, precision and reproducibility. It was shown that the method was linear over a sample concentration range of 1–100 μg/ml. Upon assessment of the intra-day and inter-day precision, the relative standard deviations (RSD) were within the range of the methodical error (approximately 5%), except at the lower concentration of 10 μg/ml, where the intra-day RSD was relatively high (17.8%). The recovery of rIL-2 upon liposome preparation and subsequent analysis of the samples was in the range 94±9%. The results indicate that the method is suitable for routine quantitation of rIL-2 in liposomal samples.     

Development of a high-performance liquid chromatographic method for bioanalytical applications with sulpiride

An improved HPLC method using a silica gel column with fluorescence detection (excitation at 300 nm and emission at 365 nm) was developed for the determination of sulpiride concentrations in plasma. Analysis of sulpiride in plasma samples was simplified by a one-step liquid–liquid extraction after alkaline treatment of only 1 ml of plasma. The low limit of quantitation was 20 ng/ml with a coefficient of variation of less than 20%. A linear range was found from 20 to 1500 ng/ml. This HPLC method was validated with the precision for inter-day and intra-day runs being 0.36–8.01% and 0.29–5.25%, respectively, and the accuracy (standard deviation of mean, SD) for inter-day and intra-day runs being −1.58 to 5.02% and −2.14 to 5.21%, respectively. Bioequivalence of the two products was evaluated in 12 normal healthy male volunteers in a single-dose, two-period, two-sequence, two-treatment cross-over study. Sulpiride plasma concentrations were analyzed with this validated HPLC method. Results demonstrated that the two tablet formulations of sulpiride appear to be bioequivalent.     

Sensitive high-performance thin-layer chromatographic method for the estimation of diospyrin, a tumour inhibitory agent from the stem bark of Diospyros montana Roxb.

Diospyrin, a tumour inhibitory agent from the stem bark of Diospyros montana was isolated and characterised. A sensitive high-performance thin-layer chromatographic (HPTLC) method was developed for the estimation of diospyrin. The method was validated for precision (intra- and inter-day), repeatability and accuracy. The method was found to be precise, with the RSDs for intra-day in the range of 0.72–1.85% and RSDs for inter-day in the range of 1.06–2.95%, for different concentrations. Instrumental precision and repeatability of the method were found to be 0.086 and 0.937 (% CV), respectively. Accuracy of the method was checked by performing the recovery study at two levels and average percentage recovery was found to be 97.87%. The developed HPTLC method was adopted for the estimation of diospyrin content of the stem bark of D. montana from different regions, which varied from 0.35 to 0.47% (w/w) in the samples.     

High-performance liquid chromatographic determination of diclofenac in human plasma using automated column switching

A validated high-performance liquid chromatographic procedure employing ultraviolet detection for the analysis of diclofenac in human plasma is reported. The method is rapid and, coupled with column switching, leads to a sensitive, accurate and reproducible assay. The retention times of diclofenac and the internal standard (4′-methoxydiclofenac, CGP-4287) are 6.4 and 7.6 min, respectively. The peak height versus plasma concentration is linear over the range 5.0–2000 ng/ml with a detection limit below 2.5 ng/ml. The mean absolute recovery of diclofenac using the described assay is 96.5% (n = 24). The inter- and intra-day accuracy and precision are within 8.3% of the actual values for all concentrations investigated. Furthermore, this procedure is applied to assess the pharmacokinetics of a single 75-mg oral dose of diclofenac sodium.     

High-performance liquid chromatographic assay for the determination of 2′-deoxy-3′-thiacytidine (lamivudine) in human plasma

A method for the quantification of 2′-deoxy-3′-thiacytidine (lamivudine, 3-TC), which incorporated the use of 3-isobutyl-methylxanthine as internal standard (I.S.) was developed and validated in human plasma, using HPLC with UV absorbance detection. Using solid-phase extraction, 3-TC and I.S. were selectively extracted from human plasma. Subsequently, chromatographic separation was performed using a YMC phenyl column with ion-pair chromatography and detection at 270 nm. The method was validated over a concentration range of 10 to 5000 ng/ml using 0.5 ml of human plasma. The extraction recovery for both 3-TC and I.S. was greater than 95%. The determination of inter- and intra-day precision (RSD) was less than 10% at all concentration levels, while the inter- and intra-day accuracy (% difference) was less than 6%.     

Quantification of fluoxetine and norfluoxetine serum levels by reversed-phase high-performance liquid chromatography with ultraviolet detection

A rapid and sensitive high-performance liquid chromatography assay method was developed to determine serum fluoxetine and norfluoxetine levels by single extraction of 0.1 ml of serum with sodium hydroxide. The mobile phase (55% acetonitrile–45% distilled water containing 10 mM aqueous triethylamine) was used to separate fluoxetine and norfluoxetine (25–1000 ng/ml, using clomipramine as the internal standard) by ultraviolet detection at 226 nm. The inter- and intra-day variabilities of fluoxetine and norfluoxetine were 13–18%, and the recoveries of both drugs exceeded 89%. This assay method was applied to a pharmacokinetic disposition study of fluoxetine in mice.     

Simple and rapid assay for acetaminophen and conjugated metabolites in low-volume serum samples

The use of marker compounds for estimating drug metabolic capacity or pharmacokinetic parameters is common in the biological sciences. Often small laboratory animals are used and thus sample size is a limiting concern. In this report, we describe an assay we developed for measuring the concentration of acetaminophen and its conjugated metabolites in low-volume serum samples. Acetaminophen and metabolites were removed from 10 μl serum samples by a single-step 6% (v/v) perchloric acid deproteination using theophylline as internal standard. Samples were separated in a pH 2.2 sodium sulfate–acetonitrile mobile phase at a flow-rate of 1.5 ml/min on a 15 cm octadecylsilyl column at room temperature. Analytes were detected at a wavelength of 254 nm. The resulting chromatograms showed no interfering peaks from endogenous serum components. The concentration ranges measured were 1.56–200 μg/ml for acetaminophen and acetaminophen sulfate and 3.91–500 μg/ml for acetaminophen glucuronide. The assay was linear in the range of concentrations analyzed. The intra-day and inter-day coefficient of variation ranged from 0.4 to 8.2% and 0.2 to 12.3% for acetaminophen, 0.5 to 12.9% and 0.3 to 16.1% for acetaminophen glucuronide, and 0.4 to 8.1% and 0.2 to 14.3% for acetaminophen sulfate, respectively. Results from the experiments show that acetaminophen and its conjugated metabolites can easily and reproducibly be measured in low-volume serum samples and thus may offer an additional method to measure these compounds when the volume of biological samples may be limited.     

High-performance liquid chromatographic assay for the α-melanotropin[4,10] fragment analogue (Melanotan-II) in rat plasma

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of Melanotan-II (MT-II), a cyclic heptapeptide which promotes rapid tanning of the skin, in rat plasma. The method involves precipitation of plasma proteins followed by direct-injection HPLC with ultraviolet detection. Calibration curves were linear over the range 100–1000 ng/ml for rat plasma. The method is reproducible and reliable with a detection limit of 50 ng/ml in plasma. Within- and between-day precision and accuracy reported as coefficient of variation and relative error, respectively, were < 7%. The application of the assay was successfully demonstrated by quantifying the concentration of MT-II in rat plasma samples following an intravenous dose of 0.3 mg/kg.     

Use of novel solid-phase extraction sorbent materials for high-performance liquid chromatography quantitation of caffeine metabolism products methylxanthines and methyluric acids in samples of biological origin

An automated reversed-phase high-performance liquid chromatographic (RP-HPLC) method, using a linear gradient elution, is described for the simultaneous analysis of caffeine and metabolites according to their elution order: 7-methyluric acid, 1-methyluric acid, 7-methylxanthine, 3-methylxanthine, 1-methylxanthine, 1,3-dimethyluric acid, theobromine, 1,7-dimethyluric acid, paraxanthine and theophylline. The analytical column, an MZ Kromasil C₄, 250×4 mm, 5 μm, was operated at ambient temperature with back pressure values of 80–110 kg/cm². The mobile phase consisted of an acetate buffer (pH 3.5)–methanol (97:3, v/v) changing to 80:20 v/v in 20 min time, delivered at a flow-rate of 1 ml/min. Paracetamol was used as internal standard at a concentration of 6.18 ng/μl. Detection was performed with a variable wavelength UV–visible detector at 275 nm, resulting in detection limits of 0.3 ng per 10-μl injection, while linearity held up to 8 ng/μl for most of analytes, except for paraxanthine and theophylline, for which it was 12 ng/μl and for caffeine for which it was 20 ng/μl. The statistical evaluation of the method was examined performing intra-day (n=6) and inter-day calibration (n=7) and was found to be satisfactory, with high accuracy and precision results. High extraction recoveries from biological matrices: blood serum and urine ranging from 84.6 to 103.0%, were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and methanol–acetate buffer (pH 3.5) (50:50, v/v) as eluent, requiring small volumes, 40 μl of blood serum and 100 μl of urine.     

Rapid determination of tetracycline antibiotics in serum by reversed-phase high-performance liquid chromatography with fluorescence detection

A rapid and accurate determination of tetracycline antibiotics in human serum by reversed-phase high-performance liquid chromatography with fluorescence detection has been developed, based on protein precipitation in serum. Various reagents for precipitation were investigated, and 24% trichloroacetic acid in methanolic solution gave the maximum recovery (at least 94.3%) and interference-free chromatograms of different three tetracyclines. At a concentration of 0.5 μg/ml, the precision (relative standard deviation) ranged from 1.12 to 1.94%. In the range 0.04–10.0 μg/ml for oxytetracycline and chlorotetracycline and 0.01–10.0 μg/ml for tetracycline, linear responses were observed. The detection limits of this method were 10–35 ng/ml for all three antibiotics. The proposed method was applied to the determination of serum concentrations in subjects receiving tetracycline antibiotics.     

High-performance liquid chromatographic assay with a simple extraction procedure for sensitive quantification of mycophenolic acid in rat and human plasma

We describe a novel sensitive and simplified gradient HPLC assay for quantification of the immunosuppressant mycophenolic acid (MPA) in rat and human plasma. In contrast to previously reported MPA assays, our method used a single step extraction comprising addition of acetonitrile, which contained phenolphthalein glucoronic acid as internal standard, for protein precipitation. Linearity: 0.1–100 μg/ml (r²>0.999), mean recoveries: MPA 98.0%, internal standard 105.2%, mean intra-day precision: 4.3%, mean day-to-day precision: 4.3%, mean day-to-day accuracy: −1.5%. Sensitivity was sufficient to allow for quantification of mycophenolic acid in as little as 50 μl plasma.     

Liquid chromatography–tandem mass spectrometry analysis of cocaine and its metabolites from blood, amniotic fluid, placental and fetal tissues: study of the metabolism and distribution of cocaine in pregnant rats

The ability to simultaneously quantitate cocaine and its 12 metabolites from pregnant rat blood, amniotic fluid, placental and fetal tissue homogenates aids in elucidating the metabolism and distribution of cocaine. An efficient extraction method was developed to simultaneously recover these 13 components using underivatized silica solid-phase extraction (SPE) cartridges. The overall recoveries for cocaine and its metabolites were studied from pregnant rat blood (47–100%), amniotic fluid (61–100%), placental homogenate (31–83%), and fetal homogenate (39–87%). Extraction of the samples using silica is not classical SPE, but rather allows for the concentration of the sample into a small volume prior to injection and the removal of the proteins due to their strong interaction with the active silica surface. A positive ion mode electrospray ionization liquid chromatography–tandem mass spectrometry (LC–MS–MS) method was used and validated to simultaneously quantitate cocaine and 12 metabolites from these four biological matrices. A gradient elution method with a Zorbax XDB C₈ reversed-phase column was used to separate the components. Multiple reaction monitoring (MRM) of a product ion arising from the corresponding precursor ion was used in order to enhance the selectivity and sensitivity of the method. Low background noise was observed from the complex biological matrices due to efficient SPE and the selectivity of the MRM mode. Linear calibration curves were generated from 0.01 to 2.50 ppm. The method also showed high intra-day (n=3) and inter-day (n=9) precision (% RSD) and accuracy (% error) for all components. The limits of detection (LODs) for the method ranged from 0.15 to 10 ppb. The LODs of cocaine and its major metabolites were less than 1 ppb from all four biological matrices. This method was applied to the study of the metabolism and distribution of cocaine in pregnant rats following intravenous infusion to a steady state plasma drug concentration. The following results were observed in the pregnant rat study: (1) the observations correlated strongly with the previous literature data on cocaine metabolism and distribution, (2) cocaine and norcocaine accumulated in the placenta, (3) arylhydroxylation of cocaine was a major metabolic pathway, (4) para-arylhydroxylation of cocaine was favored over meta-arylhydroxylation in rats and (5) accumulation of cocaine and its major metabolites was observed in the amniotic fluid.     

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml⁻¹ for gastric juice aspirate and plasma and 0.010 μg g⁻¹ for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml⁻¹ for plasma and gastric juice aspirate and up to 40 μg g⁻¹ for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

Sensitive high-performance liquid chromatographic determination of nifedipine in dog plasma using an automated sample preparation system with laboratory robot

Nifedipine, a calcium-channel blocking drug was analysed in dog plasma after oral dosing with two different formulations. Sample preparation was automated with a laboratory robot. Quantitative determination of the drug was performed on a reversed-phase HPLC system with electrochemical detection (ED) using an internal standard. Validation of the analytical method showed that the system is well suited for pharmacokinetic studies on dogs. The assay was linear in the range 1–50 ng/ml. Inter-day and intra-day variability were between 6.43–18.15% C.V. and 1.57–5.53% C.V., respectively.     

Quantification of raltegravir (MK0518) in human plasma by high-performance liquid chromatography with photodiode array detection

A precise and accurate high-performance liquid chromatography (HPLC) method with photodiode array detection has been developed and validated for raltegravir, a human immunodeficiency virus integrase strand transfer inhibitor (HIV-1 INSTI). Plasma (300 μL) was extracted with dichloromethane/hexane 50:50 (v/v) after addition of the internal standard, 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. The compounds were separated using a dC18 column and detected with ultraviolet detection at 320 nm. The limit of quantification was 10 ng/mL for raltegravir. The method was linear and validated over a concentration range of 0–10,000 ng/mL. The intra-day precision ranged from 3.1 to 12.3%, while the intra-day accuracy ranged from ?15.0 to ?0.5%, the inter-day precision and accuracy were less than 7%. The mean recovery was 76.8%. Application to clinical samples taken from patients treated with raltegravir indicated that the method is suitable for measuring plasma concentrations of raltegravir in pharmacokinetic studies of clinical trials.     

Determination of HP 749, a potential therapeutic agent for Alzheimer's disease, in plasma by high-performance liquid chromatography

N-(n-Propyl)-N-(4-pyridinyl)-1H-indol-1-amine hydrochloride (HP 749, I), a non-receptor-dependent cholinomimetic agent with noradrenergic activity, is a potential agent for the treatment of Alzheimer's disease. Pharmacokinetic studies in animals and humans showed that I was well absorbed and metabolized primarily to the N-despropyl metabolite (P7480, II) after oral administration. To facilitate the kinetic studies, a sensitive and selective high-performance chromatographic assay was developed. I and II are extracted from plasma by a mixture of cyclohexane—ethyl acetate and chromatographed on an isocratic reversed-phase high-performance liquid chromatographic system employing an analytical phenyl column with acetonitrile—ammonium formate as mobile phase. The concentrations of these two compounds, quantitated by internal standardization, are monitored by ultraviolet detection. The method is linear in the plasma assay over a concentration range of 0.5–500 ng/ml for both compounds with a quantitation limit of 0.5 ng/ml. The precision and accuracy of the calibration curves and/or method are less than 10%. The recovery of I and II from plasma is 63–74 and 63–68%, respectively, over a concentration range of 0.5–500 ng/ml.