Quantification of phenytoin and its metabolites in equine plasma and urine using high-performance liquid chromatography

A reliable and sensitive method for the extraction and quantification of phenytoin (5,5′-diphenylhydantoin), its major metabolite, 5-(p-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) in horse urine and plasma is described. The method involves the use of solid-phase extraction (SPE), liquid–liquid extraction (LLE), enzyme hydrolysis (EH) and high-performance liquid chromatography (HPLC). The minor metabolite, 5-(m-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) was not present in a reliably quantifiable concentration in all samples. The new method described was successfully applied in the pharmacokinetic studies and elimination profile of phenytoin and p-HPPH following oral or intravenous administration in the horse.     

Quantitation of a new cholecystokinin and gastrin receptor antagonist (L-365,260) in dog and rat plasma by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure has been developed for the quantification of L-365,260 (I), a cholecystokinin and gastrin receptor antagonist, in dog and rat plasma. The method involves liquid—liquid extraction and HPLC with ultraviolet detection. Standard curves were linear over the range 7.5–2000 ng/ml for rat and dog plasma. The method is reproducible and reliable with a detection limit of 7.5 ng/ml in biological fluids. The mean coefficients of variation for concentrations within the range of the standard curve range were 3.84 and 2.56%, respectively, for intra-day analysis and 4.48 and 4.26%, respectively, for inter-day analysis. Application of the development was successfully demonstrated by quantifying the concentration of I in both dog and rat plasma samples following an intravenous or oral dose of 5 mg/kg I.     

Gas chromatographic–mass spectrometric assay for rocuronium with potential for quantifying its metabolite, 17-desacetylrocuronium, in human plasma

A rapid, sensitive and selective method has been developed for the quantification of plasma concentrations of neuromuscular blocking drug, rocuronium, using gas chromatography with mass spectrometric detection. 3-Desacetylvecuronium served as the internal standard. The method involved iodide ion pair formation and a single-step liquid–liquid extraction with dicholoromethane. This method also permits simultaneous determination of its putative metabolite, 17-desacetylrocuronium, although the high detection limit for the metabolite limits the practical application of this method in pharmacokinetic study of the metabolite. The extraction efficiency was 75% for rocuronium and 50% for 17-desacetylrocuronium. The limit of quantification was 26 ng/ml for rocuronium and 870 ng/ml for its metabolite. The assay was used successfully in a patient undergoing liver transplantation and receiving rocuronium as a constant rate infusion and in a patient undergoing general elective surgery receiving the drug as an intravenous bolus. This assay is a time-saving alternative to published gas or liquid chromatographic methods for assaying rocuronium.     

Liquid chromatographic–mass spectrometric determination of itraconazole and its major metabolite, hydroxyitraconazole, in dog plasma

A fast, reliable and sensitive liquid chromatography–mass spectrometry (LC–MS) assay for the determination of itraconazole and hydroxyitraconazole in dog plasma has been developed. The analysis involves a simple liquid–liquid extraction followed by LC–MS analysis using electrospray ionization in the positive mode. Total separation of itraconazole, hydroxyitraconazole and the internal standard, miconazole, was achieved on a C₁₈ column in 3.5 min using an isocratic mixture of acetonitrile and 10 mM ammonium acetate. The response was linear over four-orders of magnitude, allowing reliable quantification of each species. This paper describes the development of the method and its validation.     

Quantification of 3,4-methylenedioxymetamphetamine and its metabolites in plasma and urine by gas chromatography with nitrogen–phosphorus detection

A gas chromatographic method with nitrogen–phosphorus detection involving a solid–liquid extraction phase was developed and validated for the simultaneous quantification of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in plasma. A modification of this method was validated for the analysis of MDMA, MDA, 4-hydroxy-3-methoxymethamphetamine (HMMA) and, 4-hydroxy-3-methoxyamphetamine (HMA) in urine. Under the analytical conditions described, the limits of detection in plasma and urine were less than 1.6 μg/l and 47 μg/l, respectively, for all the compounds studied. Good linearity was observed in the concentration range evaluated in plasma (5–400 μg/l) and urine (100–2000 μg/l) for all compounds tested. The recoveries obtained from plasma were 85.1% and 91.6% for MDMA and MDA, respectively. Urine recoveries were higher than 90% for MDMA and MDA, 74% for HMMA, and 64% for HMA. Methods have been successfully used in the assessment of plasma and urine concentrations of MDMA and its main metabolites in samples from clinical studies in healthy volunteers.     

Simultaneous determination of local anesthetics including ester-type anesthetics in human plasma and urine by gas chromatography–mass spectrometry with solid-phase extraction

The present study describes the simultaneous determination of seven different kinds of local anesthetics and one metabolite by GC–MS with solid-state extraction: Mepivacaine, propitocaine, lidocaine, procaine (an ester-type local anesthetics), cocaine, tetracaine (an ester-type local anesthetics), dibucaine (Dib) and monoethylglycinexylidide (a metabolite of lidocaine) were clearly separated from each other and simultaneously determined by GC–MS using a DB-1 open tubular column. Their recoveries ranged from 73–95% at the target concentrations of 1.00, 10.0 and 100 μg/ml in plasma, urine and water. Coefficients of variation of the recoveries ranged from 2.3–13.1% at these concentrations. The quantitation limits of the method were approximately 100 ng/ml for monoethylglycinexylidide, propitocaine, procaine, cocaine, tetracaine and dibucaine, and 50 ng/ml for lidocaine and mepivacaine. This method was applied to specimens of patients who had been treated with drip infusion of lidocaine, and revealed that simultaneous determination of lidocaine and monoethylglycinexylidide in the blood and urine was possible.     

Liquid chromatographic determination of ketoconazole, a potent inhibitor of CYP3A4-mediated metabolism

A high-performance liquid chromatographic assay with UV detection has been developed for the determination of ketoconazole in human plasma. Quantitative extraction was achieved by a single solvent extraction involving a mixture of acetonitrile–n-butyl chloride (1:4, v/v). Ketoconazole and the internal standard (clotrimazole) were separated on a column packed with Inertsil ODS-80A material and a mobile phase composed of water–acetonitrile–tetrahydrofuran–ammonium hydroxide–triethylamine (45:50.2:2.5:0.1:0.1, v/v). The column effluent was monitored at a wavelength of 206 nm with a detector range set at 0.5. The calibration graph was linear in the range of 20–2000 ng/ml, with a lower limit of quantitation of 20.0 ng/ml. The extraction recoveries for ketoconazole and clotrimazole in human plasma were 93±9.7% and 83±10.0%, respectively. The developed method has been successfully applied to a clinical study to examine the pharmacokinetics of ketoconazole in a cancer patient.     

Quantitative analysis of levamisole in porcine tissues by high-performance liquid chromatography combined with atmospheric pressure chemical ionization mass spectrometry

This work presents the development and the validation of an LC–MS–MS method with atmospheric pressure chemical ionization for the quantitative determination of levamisole, an anthelmintic for veterinary use, in porcine tissue samples. A liquid–liquid back extraction procedure using hexane–isoamylalcohol (95:5, v/v) as extraction solvent was followed by a solid-phase extraction procedure using an SCX column to clean up the tissue samples. Methyllevamisole was used as the internal standard. Chromatographic separation was achieved on a LiChrospher^® 60 RP-select B (5 μm) column using a mixture of 0.1 M ammonium acetate in water and acetonitrile as the mobile phase. The mass spectrometer was operated in MS–MS full scanning mode. The method was validated for the analysis of various porcine tissues: muscle, kidney, liver, fat and skin plus fat, according to the requirements defined by the European Community. Calibration graphs were prepared for all tissues and good linearity was achieved over the concentration ranges tested (r>0.99 and goodness of fit <10%). Limits of quantification of 5.0 ng/g were obtained for the analysis of levamisole in muscle, kidney, fat and skin plus fat tissues, and of 50.0 ng/g for liver analysis, which correspond in all cases to half the MRLs (maximum residue limits). Limits of detection ranged between 2 and 4 ng/g tissue. The within-day and between-day precisions (RSD, %) and the results for accuracy fell within the ranges specified. The method has been successfully used for the quantitative determination of levamisole in tissue samples from pigs medicated via drinking water. Moreover the product ion spectra of the levamisole peak in spiked and incurred tissue samples were in close agreement (based on ion ratio measurements) with those of standard solutions, indicating the worthiness of the described method for pure qualitative purposes.     

High-performance liquid chromatographic assay with ultraviolet detection for the determination of etoperidone and two active metabolites, 5-(1-hydroxyethyl)etoperidone and 1-(3-chlorophenyl)piperazine, in plasma

A selective and sensitive high-performance liquid chromatographic assay with ultraviolet detection for the determination of the antidepressant drug etoperidone and two active metabolites in plasma is described. The drug, metabolites and internal standard are isolated from plasma using a two-step liquid—liquid extraction procedure. The resulting sample is chromatographed on a C₁₈ column (10 cm × 2.1 mm I.D.) with ultraviolet detection at 254 nm. Standard curves are linear for each compound over the concentration range 2–1000 ng/ml. The accuracy and precision of the assay, expressed as the percentage deviation of measured values from the true value and the relative standard deviation (inter-run), are ≤ 10% at all concentrations except the minimum quantification limit. Using an automated injector and computerized data acquisition, eighty samples can be routinely processed in one day. The assay has been successfully used for the analysis of plasma samples from pharmacokinetic studies in mice, rats, dogs and humans.     

Column-switching high-performance liquid chromatographic assay for determination of asiaticoside in rat plasma and bile with ultraviolet absorbance detection

A column-switching high-performance liquid chromatography (HPLC) method is described for the determination of asiaticoside in rat plasma and bile using column-switching and ultraviolet (UV) absorbance detection. Plasma was simply deproteinated with acetonitrile prior to injection and bile was directly injected onto the HPLC system consisting of a clean-up column, a concentrating column, and an analytical column, which were connected with two six-port switching valves. Detection of asiaticoside was accurate and repeatable, with a limit of quantification of 0.125 μg/ml in plasma and 1 μg/ml in bile. The calibration curves were linear in a concentration range of 0.125–2.5 μg/ml and 1–20 μg/ml for asiaticoside in rat plasma and bile, respectively. This method has been successfully applied to determine the level of asiaticoside in rat plasma and bile samples from pharmacokinetics and biliary excretion studies.     

Sensitive and specific determination of clindamycin in human serum and bone tissue applying liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry

A method for the quantification of clindamycin in human serum and in human bone tissue samples applying high-performance liquid chromatography with atmospheric pressure chemical ionization–mass spectrometry (APCI–MS) is presented. Lincomycin is used as the internal standard. Serum samples are prepared only by protein precipitation with acetonitrile. Bone tissue samples have to be crushed and homogenized in extraction buffer prior to analysis. The chromatographic separation is achieved on an RP-18 stationary phase with 0.02% trifluoroacetic acid in water 60%/acetonitrile 40% v/v as mobile phase. The limits of quantification are 0.1 μg/ml for serum samples and 0.1 μg/g for bone tissue samples. The coefficients of variation for the assays are 4.48 and 8.41% at the limit of quantification for serum and bone tissue samples, respectively. Bone tissue samples as small as 50 mg can be used.     

Therapeutic drug monitoring of flecainide in serum using high-performance liquid chromatography and electrospray mass spectrometry

High-performance liquid chromatography with electrospray mass spectrometry (LC–MS) was used for analysis of the drug flecainide in serum. The clean-up was performed by solid-phase extraction, and an aromatic ring positional isomer was used as internal standard. Results from method validation on spiked serum samples showed excellent reproducibility; intra- and inter-assay variations (C.V.% and %Bias) were less than 6% within the therapeutic concentration range of the drug (0.2–1.0 μg/ml). Linearity was demonstrated from 0.05 to 2.0 μg/ml. The limit of detection and quantification was 0.025 and 0.05 μg/ml, respectively. Due to the high selectivity of the mass spectrometric detection, no interferences were observed. Results from clinical samples (n=18) from patients in treatment with Tambocor (flecainide acetate) showed excellent correlation with parallel data obtained from a method based on high-performance liquid chromatography (HPLC) with fluorescence detection after liquid/liquid extraction. The chromatographic separation of flecainide and internal standard was improved compared to earlier HPLC methods. The methodology is simple, accurate and requires only 0.25 ml of sample. It is a well suited method for routine therapeutic drug monitoring in a hospital or clinical chemistry laboratory.     

Determination of the tricyclic compound adosupine and its three metabolites in plasma and brain of rat using high-performance liquid chromatography

An analytical method for the detection in biological samples of the novel tricyclic compound adosupine (10-acetoamido-5-methyl-5,6-dihydro-11H-dibenzo[b,e]azepin-6,11-dione), which is capable of influencing various forms of urinary bladder hyperreflexia has been developed using high-performance liquid chromatography with UV detection. Liquid—liquid extraction was used to isolate the parent compound, three metabolites and an analogue (added as internal standard) from plasma and brain of rat. Adosupine was well separated from its three metabolites with 0.01 M disodium hydrogenphosphate—acetonitrile—methanol—nonylamine (59.986:38:2:0.014) at pH 4.5 as mobile phase using a C₁₈ reversed-phase column. The standard curves were linear in the range 50–5000 ng/ml (or ng/g) for adosupine and metabolites in both plasma and brain. The between- and within-assay variations for high and low concentrations of the parent compound and the three metabolites were 8.2–14%. In the range 50–5000 ng/ml (or ng/g) the accuracy of the method was satisfactory, with the relative error always lower than 10%. Analytical recoveries of added adosupine and the three metabolites were higher than 82%. The method has been applied successfully, to investigate the pharmacokinetics of the drug and its distribution in the central nervous system of rats.     

Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography–electrospray ionization tandem mass spectrometry

We developed highly sensitive detection of testosterone (T) and 5α-dihydrotestosterone (DHT) by liquid chromatography–electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17β-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN–MeOH–H₂O–formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3 mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.     

Determination of the glucocorticoid fluticasone propionate in plasma by automated solid-phase extraction and liquid chromatography–tandem mass spectrometry

A sensitive, robust and high throughput mass spectrometry based method is described for the determination of the glucocorticoid fluticasone propionate in plasma. The method employs solid-phase extraction in 96 well microtitre plate format which has been automated by means of a custom built Zymark robotic system. The extracts are analysed by liquid chromatography–tandem mass spectrometry using thermally and pneumatically assisted electrospray ionisation and selected reaction monitoring. The method is both accurate and precise with both intra- and inter-assay precision (C.V.) of less than <6%. The method provides a lower limit of quantification of 20 pg/ml from 0.5 ml of human plasma, sufficient to monitor systemic concentrations of inhaled fluticasone propionate at therapeutic doses.     

Determination of 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiophen-2-yl]cyclopent-3-enecarboxylic acid (CP-191,166), an angiotensin II antagonist, in dog and rat plasma by high-performance liquid chromatography

A simple and precise high-performance liquid chromatographic (HPLC) assay was developed and validated for the determination of a novel angiotensin II antagonist, 1-[5-(2-cyclopropyl-5,7-dimethyl-imidazo[4,5-b]pyridin-3-ylmethyl)thiopen-2-yl)cyclopent-3-enecarboxylic acid (CP-191,166, I), in dog and rat plasma. The internal standard (II, a saturated derivative of I) and analyte were extracted by liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Zorbax C₈ narrow-bore column with ultraviolet detection at 289 nm. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–10.0 μg/ml (r²>0.99). In dog and rat plasma, intra- and inter-assay precision ranged from 0.00 to 3.36% and 0.00 to 4.95%, respectively. The average recoveries were similar (73%) for both I and II and the upper limit of quantification of I can be as high as 500 μg/ml. The method described has been successfully applied to the quantification of I in about 2000 dog and rat plasma samples over a nine-month period.     

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.     

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.     

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.     

Validated high-performance liquid chromatographic method for the determination of lamotrigine in human plasma

A high-performance liquid chromatographic (HPLC) procedure for lamotrigine was developed and validated. Lamotrigine (LTG) and an internal standard were extracted from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 0.78–46.95 μmol/l, with a mean coefficient of correlation (r)≥0.99923. The limit of detection (LOD) and limit of quantification (LOQ) were 0.19 and 0.58 μmol/l, respectively. Within- and between-run precision studies demonstrated C.V.<3% at all tested concentrations. LTG median recovery was 86.14%. Antiepileptic drugs tested did not interfere with the assay. The method showed to be appropriate for monitoring LTG in plasma samples.