Determination of betulinic acid in mouse blood, tumor and tissue homogenates by liquid chromatography–electrospray mass spectrometry

A rapid and sensitive high-performance liquid chromatography–electrospray MS method has been developed to determine tissue distribution of betulinic acid in mice. The method involved deproteinization of these samples with 2.5 volumes (v/w) of acetonitrile–ethanol (1:1) and then 5 μl aliquots of the supernatant were injected onto a C₁₈ reversed-phase column coupled with an electrospray MS system. The mobile phase employed isocratic elution with 80% acetonitrile for 10 min; the flow-rate was 0.7 ml/min. The column effluent was analyzed by selected ion monitoring for the negative pseudo-molecular ion of betulinic acid [M−H]⁻ at m/z 455. The limit of detection for betulinic acid in biological samples by this method was approximately 1.4 pg and the coefficients of variation of the assay (intra- and inter-day) were generally low (below 9.1%). When athymic mice bearing human melanoma were treated with betulinic acid (500 mg/kg, i.p.), distribution was as follows: tumor, 452.2±261.2 μg/g; liver, 233.9±80.3 μg/g; lung, 74.8±63.7 μg/g; kidney, 95.8±122.8 μg/g; blood, 1.8±0.5 μg/ml. No interference was noted due to endogenous substances. These methods of analysis should be of value in future studies related to the development and characterization of betulinic acid.     

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.     

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.     

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans,trans-Muconic acid (1,3-butadiene-1,4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C₁₈ column (5 × 0.46 cm I.D., 3 μm particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50–500 μg/l range; the quantification limit was 6 μg/l; day-to-day precision, at 300 μg/l, C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean±S.D.=77±54 μg/l, N = 82) were statistically different from those of smoerks (169±85 μg/l, N = 30) (P<0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.     

Determination of sotalol in human cardiac tissue by high-performance liquid chromatography

A sensitive and quantitative reversed-phase HPLC method for the analysis of -sotalol in human atria, ventricles, blood and plasma was developed. Sotalol was determined in about 100 mg of human right atria, left ventricles, and in 500 μl of blood and plasma samples of patients undergoing coronary bypass surgery or heart transplantation. Patients were taking 80–160 mg of sotalol as an antiarrhythmic agent. Atenolol was used as an internal standard certifying high precision of measurement. Sotalol blood and plasma concentrations correlated linearly to the obtained signals from 26.5 ng/ml to 2.12 μg/ml. Sotalol tissue concentrations showed linearity between 0.27 ng/mg and 10.6 ng/mg wet weight. The limit of quantitation was 0.27 ng/mg at a signal-to-noise ratio of 10. Sotalol was extracted from homogenized tissue with a buffer solution (pH 9) and the remaining pellet was extracted with methanol. The methanol extract was evaporated under nitrogen and reconstituted in buffer (pH 3). The whole extract was cleaned by solid-phase column extraction, eluted with methanol, evaporated again, reconstituted in the mobile phase (acetonitrile-15 mM potassium phosphate buffer pH 3, 17:83, v/v) and injected onto the HPLC column (Spherisorb C₆ column, 5 μm,, 150×4.6 mm I.D). For the detection of sotalol, the UV wavelength was set to 230 nm. Recoveries of sotalol and atenolol in atria and ventricles were 65.6 and 75.0%, respectively. Intra- and inter-assay coefficients of variation for tissue concentrations were 3.38 and 6.14%, respectively. Intra- and inter-assay accuracy for determined tissue sotalol concentrations were 94.9±6.3 and 99.6±4.1%.     

Determination of 3-methoxy-4-hydroxyphenylethylene glycol in urine using reversed-phase liquid chromatography with column switching and electrochemical detection

A column-switching method was developed for the determination of total 3-methoxy-4-hydroxy-phenylethyleneglycol (MHPG) in urine. This was performed by first treating samples with β-glucuronidase, followed by extraction with ethyl acetate. The reconstituted extracts with injected onto an HPLC system containing an amperometric detector and tandem Nucleosil C₁₈ and C₈ reversed-phase columns connected by a switching valve. The total analysis time for MHPG was 12 min. The limit of detection was 0.18 ng, or 9 μg/l for 20-μl injections of a 1.0-ml reconstituted extract prepared from 1.0 ml of urine. The linear range extended up to 80 mg/l. The within-day precision for a urine sample containing 170 μg/l total MHPG was ±6% and the day-to-day precision was ±15%. The average levels determined by this method for total MHPG in normal subjects showed good agreement with previous literature values. This approach could be modified for the determination of free MHPG by using only ethyl acetate extraction for sample pretreatment.     

Simultaneous determination of citalopram, fluoxetine, paroxetine and their metabolites in plasma and whole blood by high-performance liquid chromatography with ultraviolet and fluorescence detection

A method for the simultaneous determination of the three selective serotonin reuptake inhibitors (SSRIs) citalopram, fluoxetine, paroxetine and their metabolites in whole blood and plasma was developed. Sample clean-up and separation were achieved using a solid-phase extraction method with C₈ non-endcapped columns followed by reversed-phase high-performance liquid chromatography with fluorescence and ultraviolet detection. The robustness of the solid-phase extraction method was tested for citalopram, fluoxetine, paroxetine, Cl-citalopram and the internal standard, protriptyline, using a fractional factorial design with nine factors at two levels. The fractional factorial design showed two significant effects for paroxetine in whole blood. The robustness testing for citalopram, fluoxetine, Cl-citalopram and the internal standard revealed no significant main effects in whole blood and plasma. The optimization and the robustness of the high-performance liquid chromatographic separation were investigated with regard to pH and relative amount of acetonitrile in the mobile phase by a central composite design circumscribed. No alteration in the elution order and no significant change in resolution for a deviation of ±1% acetonitrile and ±0.3 pH units from the specified conditions were observed. The method was validated for the concentration range 0.050–5.0 μmol/l with fluorescence detection and 0.12–5.0 μmol/l with ultraviolet detection. The limits of quantitation were 0.025 μmol/l for citalopram and paroxetine, 0.050 μmol/l for desmethyl citalopram, di-desmethyl citalopram and citalopram-N-oxide, 0.12 μmol/l for the paroxetine metabolites by fluorescence detection, and 0.10 μmol/l for fluoxetine and norfluoxetine by ultraviolet detection. Relative standard deviations for the within-day and between-day precision were in the ranges 1.4–10.6% and 3.1–20.3%, respectively. Recoveries were in the 63–114% range for citalopram, fluoxetine and paroxetine, and in the 38–95% range for the metabolites. The method has been used for the analysis of whole blood and plasma samples from SSRI-exposed patients and forensic cases.     

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.     

Simple high-performance liquid chromatography determination of ampicillin in human serum using solid-phase extraction disk cartridges

A simple and reproducible method for the analysis of ampicillin in human serum was developed. Serum samples were extracted using solid-phase extraction disk cartridges containing a sorbent of styrene divinyl/benzene. Extracts were separated by reversed-phase C₁₈ high-performance liquid chromatography with UV detection at 220 nm. The mobile phase consisted of acetonitrile–10 mM NaH₂PO₄ (6.5:93.5, v/v). Using this extraction procedure, recovery from serum was 98.4±5.6%. The quantitation limit was 0.19 μg/ml using 0.5 ml of serum. The calibration curves from 0.19 to 9.41 μg/ml were linear with correlation coefficients of 0.999. This method is suitable for therapeutic drug monitoring of ampicillin (ABPC) after oral administration of lenampicillin hydrochloride.     

Solid-phase extraction and high-performance liquid chromatographic determination of tamoxifen and its major metabolites in plasma

Tamoxifen (TAM) is a triphenylethylene anti-oestrogen, commonly used in the treatment of breast cancer. Patients receiving tamoxifen therapy may experience both de novo and acquired resistance. As one of the mechanisms for this may be extensive peripheral bio-transformation of tamoxifen, there has been considerable interest in the pharmacokinetics and metabolism of tamoxifen. A reversed-phase high-performance liquid chromatography separation has been developed to determine the levels of tamoxifen and its major metabolites in human plasma. The method is highly sensitive (2 ng/ml) and selective for tamoxifen, cis-tamoxifen (CIS), 4-hydroxytamoxifen (4-OH) and desmethyltamoxifen (DMT). A μBondapak C₁₈ 10 μm column (30 cm × 3.9 mm I.D.) was used, with a mobile phase of methanol-1% triethylamine at pH 8 (89:11, v/v). Sample preparation was carried out using a C₂ (500 mg sorbent, 3 ml reservoirs) solid phase extraction method, and extraction efficiencies were approximately 60% for TAM and its metabolites. Accuracy and precision, as determined by spiking plasma samples with a mixture of tamoxifen and its metabolites, ranged from 85–110% (± 5–10%) at 1 μg/ml, 101–118% (± 8–20%) at 0.1 μg/ml and 111–168% (± 43–63%) at 0.01 μg/ml. Results from 59 patients show mean values of 54 ng/ml for 4-OH; 190 ng/ml for DMT; 93 ng/ml for TAM and 30 ng/ml for CIS (detected in three patients only). This methodology can be applied routinely to the determination of TAM and its metabolites in plasma from patients undergoing therapy.     

Purge-and-trap gas chromatographic determination of styrene in urine and blood: Application to exposed workers

A simple purge-and-trap gas chromatographic method with flame ionization detection was developed for the determination of styrene in urine and blood. Styrene present in a 5 ml sample at room temperature was swept by helium at 40 ml/min for 11 min, trapped on a Tenax trap, desorbed by heating, cryofocused, and injected by flash heating into a DB-5 capillary GC column. The oven temperature program was from 80°C, held for 8 min, to 120°C at 5°C/min, and then held for 2 min. The detector temperature was 250°C. The calibration curves were linear in the range of 2.5–15 ppb styrene in urine and 25–150 ppb in blood. The detection limits calculated were 0.4 μg/l in urine and 0.6 μg/l in blood. The coefficients of variations within the day and day-to-day were 3 and 3.1%, respectively, for 2.5 ppb of styrene in urine, and 1 and 1.6% for 25 ppb of styrene in blood. The results obtained from samples taken from workers exposed to styrene were reported.     

Determination of pyrazinamide and its main metabolites in rat urine by high-performance liquid chromatography

A new high-performance liquid chromatograhic procedure for simultaneous determination of pyrazinamide (PZA) and its three metabolites 5-hydroxypyrazinamide (5-OH-PZA), pyrazinoic acid (PA), and 5-hydroxypyrazinoic acid (5-OH-PA), in rat urine was developed. 5-OH-PZA and 5-OH-PA standards were obtained by enzymatic synthesis (xanthine oxidase) and checked by HPLC and GC–MS. Chromatographic separation was achieved in 0.01 M KH₂PO₄ (pH 5.2), circulating at 0.9 ml/min, on a C₁₈ silica column, at 22°C. The limits of detection were 300 μg/l for PZA, 125 μg/l for PA, 90 μg/l for 5-OH-PZA and 70 μg/l for 5-OH-PA. Good linearity (r²>0.99) was observed within the calibration ranges studied: 0.375–7.50 mg/l for PZA, 0.416–3.33 mg/l for PA, 0.830–6.64 mg/l for 5-OH-PZA and 2.83–22.6 mg/l for 5-OHPA. Accuracy was always lower than ±10.8%. Precision was in the range 0.33–5.7%. The method will constitute a useful tool for studies on the influence of drug interactions in tuberculosis treatment.     

Determination of temozolomide in human plasma and urine by high-performance liquid chromatography after solid-phase extraction

As a part of a pilot clinical study, a high-performance reversed-phase liquid chromatography analysis was developed to quantify temozolomide in plasma and urine of patients undergoing a chemotherapy cycle with temozolomide. All samples were immediately stabilized with 1 M HCl (1 + 10 of biological sample), frozen and stored at −20°C prior to analysis. The clean-up procedure involved a solid-phase extraction (SPE) of clinical sample (100 μl) on a 100-mg C₁₈-endcapped cartridge. Matrix components were eliminated with 750 μl of 0.5% acetic acid (AcOH). Temozolomide was subsequently eluted with 1250 μl of methanol (MeOH). The resulting eluate was evaporated under nitrogen at RT and reconstituted in 200 μl of 0.5% AcOH and subjected to HPLC analysis on an ODS-column (MeOH-0.5% AcOH, 10:90) with UV detection at 330 nm. The calibration curves were linear over the concentration range 0.4–20 μg/ml and 2–150 μg/ml for plasma and urine, respectively. THe extraction recovery of temozolomide was 86–90% from plasma and 103–105% from urine over the range of concentrations considered. The stability of temozolomide was studied in vitro in buffered solutions at RT, and in plasma and urine at 37°C. An acidic pH (<5–6) shoul be maintained throughout the collection, the processing and the analysis of the sample to preserve the integrity of the drug. The method reported here was validated for use in a clinical study of temozolomide for the treatment of metastatic melanoma and high grade glioma.     

Determination of flumequine and 7-hydroxyflumequine in plasma of sheep by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of flumequine and its metabolite 7-hydroxyflumequine in sheep plasma was described. The two compounds were extracted from 100 μl of plasma by liquid–liquid extraction. Aliquots (100 μl) were injected onto the HPLC system and separated on a LiChrospher Select B column with an isocratic system. The compounds were detected by fluorimetric detection for concentrations below 500 μg/l and by UV detection for the concentrations exceeding 500 μg/l. The range of the validated concentrations were 50 000 to 5 μg/l and 500 to 10 μg/l with mean recovery rates of 87±3% and 60±1% for flumequine and 7-hydroxyflumequine, respectively.     

High-performance liquid chromatographic analysis of amoxicillin in human and chinchilla plasma, middle ear fluid and whole blood

We extended the application of a sensitive high-performance liquid chromatography assay of amoxicillin developed in this laboratory for human plasma and middle ear fluid (MEF) to other sample matrices including chinchilla plasma or MEF and human and chinchilla whole blood with minor modification and validated the limit of quantitation at 0.25 μg/ml with a 50-μl sample size for human and chinchilla plasmas or MEFs. Amoxicillin and cefadroxil, the internal standard, were extracted from 50 μl of the samples with Bond Elut C₁₈ cartridges. The extract was analyzed on a Keystone MOS Hypersil-1 (C₈) column with UV detection at 210 nm. The mobile phase was 6% acetonitrile in 5 mM phosphate buffer, pH 6.5 and 5 mM tetrabutylammonium. The within-day coefficients of variation were 2.7–9.9 (n=4) and 1.7–7.2% (n=3) for chinchilla plasma and MEF samples, respectively; 2.8–8.1% (n=3) and 2.9–4.7% (n=3) for human and chinchilla whole blood, respectively. An alternative mobile phase composition for chinchilla plasma and MEF samples reduced the analysis time significantly.     

Development, validation and application of assays to quantify metrifonate and 2,2-dichlorovinyl dimethylphosphate in human body fluids

Gas chromatographic procedures [GC with electron-capture detection (ECD) and GC–MS] for the quantitative analysis of metrifonate and its active metabolite 2,2-dichlorovinyl dimethylphosphate (DDVP) in human blood and urine were developed, validated, and applied to the analysis of clinical study samples. Analysis of metrifonate involved extraction of acidified blood with ethyl acetate followed by solid-phase clean-up of the organic extract. Acidified urine was extracted with dichloromethane and the residue of evaporated organic phase was reconstituted in toluene. ECD and diethyl analogue of metrifonate internal standard (I.S.) were used for quantitation of metrifonate. The metrifonate lower limit of quantitation (LOQ) was 10.0 μg/l. The DDVP metabolite was chromatographed separately after cyclohexane extraction of acidified blood and urine using d₆-DDVP I.S. and MS detection. The LOQ of DDVP was 1 μg/l. Stability studies have confirmed that the matrix should be acidified prior to storage at −20°C or −80°C to inhibit chemical and enzymatic degradation of the analytes and to avoid overestimation of DDVP concentrations. Metrifonate was found to be stable in acidified human blood after 20 months of storage at −20°C and after 23 months of storage at −80°C. Under these conditions DDVP was found to be stable after 12 months of storage. Both assay procedures were cross-validated by different world-wide laboratories and found to be accurate and robust during analyses of clinical study samples.     

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid–liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 μl of mobile phase and 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10–9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 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 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.     

Determination of anticancer drug vitamin K3 in plasma by high-performance liquid chromatography

Synthetic vitamin K₃ (VK₃, 2-methyl-1,4-naphthoquinone, or menadione) has been found to exhibit antitumor activity against various human cancer cells at relative high dose. Parallel to our study on the mechanism of VK₃ action and for future clinical trials in Taiwan, we developed a simple, sensitive and accurate high-performance liquid chromatographic method for the determination of VK₃ in biological fluids. VK₃ was extracted from the plasma samples with n-hexane. The chromatographic separation employed an ODS analytical column (5 μm, 250 × 4.6 mm I.D.) with a mobile phase of methanol-water (70:30 v/v) and UV detection at 265 nm. On completely drying of the extraction solution, n-hexane, by a stream of nitrogen, menadione was lost to a great extent. Methanol (70%, 200 μl) was added to the extraction solvent after extraction and centrifugation to prevent the loss of menadione. The absolute recovery was 82.4±7.69% (n = 7). The within-day and between-day calibration curves of VK₃ in plasma in the ranges of interest (0.01–10.00 μg/ml; 0.01–5.00 μg/ml) showed good linearity (r>0.999) and acceptable precision. The limit of quantitation of VK₃ was 10 ng/ml) showed good method has been succesfully applied to a pilot pharmacokinetic study of VK₃ in rabbits receiving an intravenous high-dose bolus injection of 75 mg menadiol sodium diphosphate (Synkayvite). The pharmacokinetic properties of menadione could be described adequately by an open two-compartment model. The mean half-life of menadiol (transformation to menadione) was 2.60±0.12 min. The elimination half-life, volume of distribution and plasma clearance of menadione were 26.3±2.97 min, 25.7±0.78 1, and 0.68±0.10 1/min, respectively.     

Analysis of oxazepam in urine using solid-phase extraction and high-performance liquid chromatography with fluorescence detection by post-column derivatization

A reversed-phase high-performance liquid chromatographic method for oxazepam in human urine samples has been developed. The sample preparation consists of an enzymatic hydrolysis with β-glucuronidase, followed by a solid-phase extraction process using Bond-Elut C₂ cartridges. The mobile phase used was a methanol—water (60:40, v/v) mixture at a flow-rate of 0.50 ml/min. The column was a 3.5 cm × 4.6 mm I.D. C₁₈ reversed-phase column. The detection system was based on a fluorescence post-column derivatization of oxazepam in mixtures of methanol and acetic acid. A linear range from 0.01 to 1 μg/ml of urine and a limit of detection of 4 ng/ml of urine were attained. Within-day recoveries and reproducibilities from urine samples spiked with 0.2 and 0.02 μg/ml oxazepam were 97.9 and 95.0 and 2.1 and 9.4%, respectively.