High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

Achiral and chiral high-performance liquid chromatographic methods for clinafloxacin, a fluoroquinolone antibacterial, in human plasma

Achiral and chiral HPLC methods were developed for clinafloxacin, a quinolone antimicrobial agent. For achiral assay, analytes were isolated from plasma by precipitating plasma proteins. Separation was achieved on a C₁₈ column using an isocratic eluent of ion pairing solution–acetonitrile (80:20, v/v) at 1.0 ml/min with UV detection at 340 nm. The ion pairing solution was 0.05 M citric acid, 1.15 mM tetrabutylammonium hydroxide and 0.1% ammonium perchlorate. Inter-assay accuracy was within 4.9% with an inter-assay precision of 3.7% over a quantitation range of 0.025 to 10.0 μg/ml. For chiral assay, analytes were isolated from plasma by solid-phase extraction. Separation was achieved on a Crownpak CR(+) column using an isocratic eluent of water–methanol (88:12, v/v) containing 0.1 mM decylamine at 1.0 ml/min with UV detection at 340 nm. Perchloric acid was added to adjust pH to 2. Inter-assay accuracy was within 3.5% with a inter-assay precision of 5.4% over a quantitation range of 0.040 to 2.5 μg/ml.     

Sensitive and convenient high-performance liquid chromatographic method for the determination of mitomycin C in human plasma

An improved high-performance liquid chromatographic assay for the cytostatic drug mitomycin C in plasma is presented. The principal steps are precipitation of plasma proteins with acetonitrile, lyophilization of the supernatant and reversed-phase chromatography on a Hypersil ODS 5 μm column with 0.01 M NaH₂PO₄ buffer (pH 6.5)-methanol (70:30, v/v) in isocratic mode. At a flow-rate of 1.3 ml/min a column pressure of 180–220 bar resulted. Porfiromycin served as internal standard. UV detection was performed at 365 nm. Quantitation limit based on a coefficient of variation <10% in intra- and inter-day assay was 5 μg/l mitomycin C, detection limit based on a signal-to-noise ratio of 3 was 1 μg/l. Recovery was 100% and linearity was shown for the whole range of concentration (1–500 μg/l). None of the five drugs used during chemoembolisation interfered with the assay in vitro. The assay meets the requirements for pharmacokinetic studies of mitomycin C in patients as regards sensitivity and ease of use.     

Determination of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma by reversed-phase liquid chromatography with ultraviolet detection

A simple and sensitive high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantitation of risperidone and its major metabolite 9-hydroxyrisperidone in human plasma, using clozapine as internal standard. After sample alkalinization with 1 ml of NaOH (2 M) the test compounds were extracted from plasma using diisopropyl ether–isoamylalcohol (99:1, v/v). The organic phase was back-extracted with 150 μl potassium phosphate (0.1 M, pH 2.2) and 60 μl of the acid solution was injected into a C₁₈ BDS Hypersil analytical column (3 μm, 100×4.6 mm I.D.). The mobile phase consisted of phosphate buffer (0.05 M, pH 3.7 with 25% H₃PO₄)–acetonitrile (70:30, v/v), and was delivered at a flow-rate of 1.0 ml/min. The peaks were detected using a UV detector set at 278 nm and the total time for a chromatographic separation was about 4 min. The method was validated for the concentration range 5–100 ng/ml. Mean recoveries were 98.0% for risperidone and 83.5% for 9-hydroxyrisperidone. Intra- and inter-day relative standard deviations were less than 11% for both compounds, while accuracy, expressed as percent error, ranged from 1.6 to 25%. The limit of quantitation was 2 ng/ml for both analytes. The method shows good specificity with respect to commonly prescribed psychotropic drugs, and it has successfully been applied for pharmacokinetic studies and therapeutic drug monitoring.     

High-performance liquid chromatographic determination of sotalol in biological fluids

A sensitive, selective and reproducible reversed-phase high-performance liquid chromatographic method is described for the quantification of sotalol in human serum and urine. Sotalol and the internal standard, atenolol, were extracted from alkalinized serum and urine (pH 9.0) into 1-butanol—chloroform (20:60, v/v). The organic phase was evaporated, and to the residue was added 0.1 M sulphuric acid (serum analysis) or mobile phase (urie analysis). The mobile phase consisted of 0.01 M phosphate buffer (pH 3.2) and acetonitrile (20:80, v/v) containing 3 mM n-octylsodium sulphate. The flow-rate was 1.5 ml/min. The retention times of atenolol and sotalol were 7 and 10 min, respectively. Ultraviolet detection at 226 nm made it possible to achieve a detection limit of 0.03 μmol/l.     

Liquid chromatographic determination of urinary 2-thiothiazolidine-4-carboxylic acid, a biomarker of carbon disulphide exposure

An effective gradient high-performance liquid chromatographic method for baseline separation of urinary 2-thiothiazolidine-4-carboxylic acid (TTCA), with photodiode array detection at 271 nm was described. o-Methylhippuric acid was used as an internal standard (I.S.). A 1-ml urine sample was saturated with 300 mg of sodium sulphate, acidified with 100 μl of 6 M hydrochloric acid, extracted twice with 2 ml of diethyl ether, and after evaporation, the residue was taken up in 1 ml of 0.1% (v/v) phosphoric acid. The two mobile phases used for gradient elution were: (A) 10 mM ammonium dihydrogenphosphate (pH 3.5) and (B) same concentration of buffer but containing 20% (v/v) of methanol (pH 4.8). The flow-rate was set at 1.0 ml/min. TTCA and I.S. were detected at 2.2 and 9.1 min, respectively. The method was validated with urine samples collected from normal subjects and workers occupationally exposed to carbon disulphide. The present method enables the detection of urinary TTCA at a concentration of 0.025 mg/l. Analytical recovery and reproducibility generally exceeded 90%. The proposed method is considered more sensitive, specific and reliable than other existing methods.     

Application of a high-performance liquid chromatographic assay for the neuromuscular blocker gallamine to analysis of rat plasma, muscle and microdialysate samples

A reliable high-performance liquid chromatographic method has been validated for determination of gallamine in rat plasma, muscle tissue and microdialysate samples. A C₁₈ reversed-phase column with mobile phase of methanol and water containing 12.5 mM tetrabutyl ammonium (TBA) hydrogen sulphate (22:78, v/v) was used. The flow-rate was 1 ml/min with UV detection at 229 nm. Sample preparation involved protein precipitation with acetonitrile for plasma and muscle tissue homogenate samples. Microdialysate samples were injected into the HPLC system without any sample preparation. Intra-day and inter-day accuracy and precision of the assay were <13%. The limit of quantification was 1 μg/ml for plasma, 1.6 μg/g for muscle tissue and 0.5 μg/ml for microdialysate samples. The assay was applied successfully to analysis of samples obtained from a pharmacokinetic study in rats using the microdialysis technique.     

Direct liquid chromatographic micro-measurement of tamoxifen in plasma of cancer patients

We describe in this report a sensitive and direct method for the analysis of tamoxifen (TAM) in microsamples of plasma. The drug and internal standard (quinine bisulfate, I.S.) were separated on a 10-μm particle, 10 cm × 8 mm CN cartridge in conjuction with a radial compression system. The mobile phase was a mixture of 0.1 M sodium acetate in 0.001 M tetrabutylammonium phosphate solution (pH 6) and methanol (30:70, v/v) at a flow-rate of 4 ml/min. After addition of I.S. and o-phosphoric acid in acetonitrile (0.6 M) to the plasma (30 μl), the mixture was placed in an ultraviolet shortwave transluminator for 2 min prior to injection into the chromatograph. The compounds were detected in the effluent fluorometrically at excitation and emission wavelengths of 258 and 378 nm, respectively. Under these conditions, no interference in the assay from any endogenous substance or other concurrently used drugs was observed and the retention times of I.S. and TAM were 4.4 and 10.15 min, respectively. The concentration of TAM in plasma was linearly (r>0.9983) related to the peak height ratio (TAM/I.S.) in the range 0.01–2.0 μg ml⁻¹ and C.V. at 0.075, 0.4 and 1.2 μg ml⁻¹ was 4.96%. We are currently using this assay for monitoring TAM in plasma and investigating its pharmacokinetics in cancer patients receiving cytotoxic drugs in addition to TAM as a multi-drug resistance modifier.     

Determination of gabapentin in plasma by high-performance liquid chromatography

A rapid and simple method for determination of the novel antiepileptic compound gabapentin [1-(aminomethyl)cyclohexaneacetic acid] in plasma is described. Blank human plasma was spiked with gabapentin (1.0–10.0 μg/ml) and internal standard [1-(aminomethyl)-cycloheptaneacetic acid; 5.0 μg/ml]. Individual samples were treated with 2 M perchloric acid, centrifuged and then derivatised with o-phthalaldehyde-3-mercaptopropionic acid. Separation was achieved on a Beckman Ultrasphere 5 μm reversed-phase column with mobile phase consisting of 0.33 M acetate buffer (pH 3.7; containing 100 mg/l EDTA)-methanol-acetonitrile (40:30:30, v/v). Eluents were monitored by fluorescence spectroscopy with excitation and emission wavelengths of 330 and 440 nm, respectively. The calibration curve for gabapentin in plasma was linear (r=0.9997) over the concentration range 1.0–10.0 μg/ml. Recovery was seen to be 90%. The inter- and intra-assay variations for three different gabapentin concentrations were 10% throughout. The lower limit of quantitation was found to be 0.5 μg/ml. Chromatography was unaffacted by a range of commonly employed antiepileptic drugs or selected amino acids.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 × 2.1 mm I.D., 5 μm Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate—acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 μl sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean ± S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 ± 1.2% and 91.4 ± 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Small blood volumes from children for quantitative sotalol determination using high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method using fluorescence detection has been developed for sotalol determination in small plasma samples of children and newborns with limited blood volume. In sample sizes of 100 μl of plasma, sotalol was extracted using an internal standard and solid-phase extraction columns. Chromatographic separation was performed on a Spherisorb C₆ column of 150×4.6 mm I.D. and 5 μm particle size at ambient temperature. The mobile phase consisted of acetonitrile–15 mM potassium phosphate buffer (pH 3.0) (70:30, v/v). The excitation wavelength was set at 235 nm, emission at 300 nm. The flow-rate was 1 ml/min. Sotalol and the internal standard atenolol showed recoveries of 107±8.9 and 97±8.1%, respectively. The linearity range for sotalol was between 0.07 and 5.75 μg/ml, the limit of quantitation 0.09 μg/ml. Precision values expressed as percent relative standard deviation of intra-assay varied between 0.6 and 13.6%, that of inter-assay between 2.4 and 14.4%. Accuracy varied between 86.1 and 109.8% (intra-assay) and 95.4 and 103.3% (inter-assay). Other clinically used antiarrhythmic drugs did not interfere. As an application of the assay, sotalol plasma concentrations in a 6-year-old child with supraventricular tachycardia treated with oral sotalol (3.2 mg/kg per day) are reported.     

Rapid and sensitive determination of paclitaxel in mouse plasma by high-performance liquid chromatography

This report describes a rapid, simple and sensitive isocratic high-performance liquid chromatography with diode array UV detection for micro-sample analysis of paclitaxel in mouse plasma. The analysis utilized a Capcell-pak octadecyl analytical column and a mobile phase consisting of acetonitrile–0.1% phosphoric acid in deionized water (55:45, v/v). Paclitaxel and n-hexyl p-hydroxybenzoic acid (internal standard) were extracted from plasma by one-step extraction with tert.-butyl methyl ether. Peak purity was determined over a UV wavelength range of 200 to 400 nm. Paclitaxel and the internal standard were eluted at 3.4 min and 5.4 min, respectively, at a mobile phase flow-rate of 1.3 ml/min. No interfering peaks were observed and the total run time was 10 min. The standard curve was linear (r=0.9999) over the concentration range of 0.010–500 μg/ml. The extraction recovery was >90% for both paclitaxel and n-hexyl p-hydroxybenzoic acid. The intra- and inter-day assay variabilities of paclitaxel ranged from 0.4 to 2.2% and 0.6 to 7.8%, respectively. The LOD and LOQ were 5 and 10 ng/ml, respectively, for paclitaxel using a plasma sample volume of 100 μl. This highly sensitive and simple assay method was successfully applied to a pharmacokinetic study after i.v. administration of paclitaxel 20 mg/kg to mice.     

Automated solid-phase extraction method for the determination of atovaquone in capillary blood applied onto sampling paper by rapid high-performance liquid chromatography

A bioanalytical method for the determination of atovaquone in 100 μl blood-spots by solid-phase extraction and high-performance liquid chromatography has been developed and validated. Atovaquone was extracted from the sampling paper in 0.2 M phosphoric acid and a structurally similar internal standard was added with acetonitrile before being loaded onto a C8 end-capped solid-phase extraction column. Atovaquone and internal standard were analysed by high-performance liquid chromatography on a C₁₈ J’Sphere ODS-M80 (150×4.0 mm) column with mobile phase acetonitrile–phosphate buffer, 0.01 M, pH 7.0 (65:35, v/v) and UV detection at 277 nm. The intra-assay precision was 2.7% at 12.00 μM and 13.5% at 1.00 μM. The inter-assay precision was 3.3% at 12.00 μM and 15.6% at 1.00 μM. The lower limit of quantification was 1.00 μM. The limit of detection was 0.50 μM.     

Determination of zolpidem in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 μl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C₁₈ column, 5 μm particle size with an isocratic mobile phase consisting of methanol–acetonitrile–26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.     

High-performance liquid chromatographic determination of the antifungal drug fluconazole in plasma and saliva of human immunodeficiency virus-infected patients

A high-performance liquid chromatographic (HPLC) assay has been developed for the determination of the antifungal drug fluconazole in saliva and plasma of patients infected with the human immunodeficiency virus (HIV). Samples can be heated at 60°C for 30 min to inactivate the virus without loss of the analyte. The sample pretreatment involves a liquid-liquid extraction with chloroform-1-propanol (4:1, v/v). The chromatographic analysis is performed on a Lichrosorb RP-18 (5 μm) column by isocratic elution with a mobile phase of 0.01 M acetate buffer (pH 5.0)-methanol (70:30, v/v) and ultraviolet (UV) detection at 261 nm. The lower limit of is 100 ng/ml in plasma (using 500-μl samples) and 1 μg/ml in saliva (using 250-μl samples) and the method is linear up to 100 μg/ml in plasma and saliva. At a concentration of 5 μg/ml the within-day and between-day precision in plasma are 7.1 and 5.7%, respectively. In saliva the within-day and between-day precision is 10.8% (at 5 μg/ml). The methodology is now being used in pharmacokinetic studies in HIV-infected patients in our hospital.     

High-performance liquid chromatographic procedure for the quantitative determination of paclitaxel (Taxol) in human plasma

An isocratic high-performance liquid chromatographic method has been developed and validated for the quantitative determination of paclitaxel (Taxol^®), a novel antimitotic, anticancer agent, in human plasma. The analysis required 0.5 ml of plasma, and was accomplished by detection of the UV absorbance of paclitaxel at 227 nm following extraction and concentration. The method involved extraction of paclitaxel from plasma, buffered with 0.5 ml of 0.2 M ammonium acetate (pH 5.0), onto 1-ml cyano Bond Elut columns. The eluent was evaporated under nitrogen and low heat, and reconstituted with the mobile phase, acetonitrile-methanol-water (4:1:5, v/v/v) containing 0.01 M ammonium acetate (pH 5.0). The samples were chromatographed on a reversed-phase octyl 5 μm column. The retention time of paclitaxel was 10 min. The validated quantitation range of the method was 10–1000 ng/ml (0.012–1.17 μM) of paclitaxel in plasma. Standard curve correlation coefficients of 0.995 or greater were obtained during validation experiments and analysis of clinical study samples. The observed recovery for paclitaxel was 83%. Epitaxol, a biologically active stereoisomer, and baccatin III, a degradation product, were also chromatographically separated from taxol by this assay. The method was applied to samples from a clinical study of paclitaxel in cancer patients, providing a pharmacokinetic profiling of paclitaxel.     

Improvement in the high-performance liquid chromatography malondialdehyde level determination in normal human plasma

We report a very rapid and simple isocratic reversed-phase HPLC separation of malondialdehyde (MDA) in normal human plasma without previous purification of the MDA–2-thiobarbituric acid (TBA) complex. The separation of MDA–TBA complex was performed using a 250×4.6 mm Nucleosil-5C18 column with a mobile phase composed of 35% methanol and 65% 50 mM sodium phosphate buffer, pH 7.0. Samples of 50 μl (composed of 100 μl plasma mixed with 1.0 ml of 0.2% 2-thiobarbituric acid in 2 M sodium acetate buffer containing 1 mM diethylenetriaminepentaacetic acid, pH 3.5, and 10 μl of 5% 2,6-di-tert.-butyl-4-methylphenol in 96% ethanol, incubated at 95°C for 45 min [K. Fukunaga, K. Takama and T. Suzuki, Anal. Biochem., 230 (1995) 20] were injected into the column. The MDA–TBA complex was eluted at a flow-rate of 1 ml/min and monitored by fluorescence detection with excitation at 515 nm and emission at 553 nm. Analysis of groups of normal male and female volunteers gave plasma levels of MDA of 1.076 nmol/ml with a coefficient of variation of about 58%. No significant statistical differences were found between male and female groups, and no correlation was discovered on the age.     

High-performance liquid chromatographic assay for melanotan-1 ([Nle-DPhe]α-melanocyte-stimulating hormone) in biological matrices

The overall objective of this research was to develop a sensitive, specific, and stability-indicating HPLC assay for the determination of the [Nle⁴-DPhe⁷]α-melanocyte-stimulating hormone analog known as Melanotan-1 (MT-1) in biological matrices, i.e., cell culture transport media and human plasma. Separation was accomplished isocratically within 8.0 min using a C₈ reversed-phase column. The mobile phase consisted of 0.1 M phosphate buffer-acetonitrile (80:20, v/v) with 18 μl/l triethylamine at pH 2.50. The flow-rate was 1 ml/min with detection at 214 nm. Standard curves (n = 5) were linear over the concentration range 100–1000 ng/ml. The precision, accuracy, intra- and inter-day variations were good with C.V.s typically within 8.7% for concentrations greater than 100 ng/ml. This method was applied to a study of the transport of MT-1 in the Caco-2 cell monolayer model.     

Determination of the angiotensin-converting enzyme inhibitor lisinopril in urine using solid-phase extraction and reversed-phase high-performance liquid chromatography

A simple, accurate and precise high-performance liquid chromatographic method is described for assaying lisinopril in human urine. Urine (1 ml) containing lisinopril and enalaprilat (internal standard) was acidified with 10 μl of 6 M nitric acid, passed through a Sep-Pak C₁₈ cartridge and eluted with 3 ml of 10% acetonitrile, followed by 6 ml of distilled water. The separations were carried out using a μBondapak C₁₈ column with a mobile phase comprising acetonitrile (60 ml), methanol (10 ml) and tetrahydrofuran (10 ml) in 15 mM phosphate buffer (920 ml) at pH 2.90. Separations were performed at 40°C and detection was at 206 nm. Standard calibration plots of lisinopril in urine were linear (r> 0.998) and recovery was greater than 64%. The lowest quantifiable concentration was 0.5 μg/ml. Within-day and between-day imprecision (coefficient of variation) ranged from 2.51% to 9.26%, and inaccuracy was less than 8.3%.