Sensitive high-performance liquid chromatographic assay for pilocarpine in biological fluids using fluorescence derivatisation

A sensitive assay for pilocarpine in biological fluids has been developed involving HPLC of a fluorescent derivative of 4-bromomethyl-7-methoxycoumarin. Pilosine as internal standard was added before the derivatisation step. The fluorescent derivatives were well resolved and separated from excess reagent and endogeneous compounds on a cyanopropyl silica column. The detection limit of pilocarpine in biological fluids was 1.0 ng/ml and the assay was linear up to a concentration of 150 ng/ml. The assay was applied to a preliminary study of pilocarpine disposition in man after a single oral dose. This is the first report of pilocarpine excretion into saliva.     

Sensitive chiral high-performance liquid chromatographic assay for labetalol in biological fluids

The four stereoisomers of the combined α- and β-adrenoceptor antagonist labetalol were separated and quantified at therapeutic concentrations by normal-phase high-pressure liquid chromatography using a chiral stationary phase and fluorescence detection. Drug in plasma or urine was recovered by solid-phase extraction with 83±5% efficiency. Limits of detection from biological samples (3 ml) were between 1.5–1.8 ng ml⁻¹. Intra-day and inter-day variation at 25 ng ml⁻¹ were ≤2.7% and ≤5.80% respectively for all stereoisomers. The assay was applied to an examination of the disposition of labetalol stereoisomers after a single oral dose of racemate to a human volunteer. Labetalol appears to undergo enantioselective metabolism leading to relatively low plasma concentrations of the pharmacologically active enantiomers.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Sensitive high-performance liquid chromatographic method using coulometric electrode array detection for measurement of phytoestrogens in dried blood spots

As the epidemiological and physiological investigation of isoflavones and lignans expands, the need for sensitive methods for analyzing large numbers of samples intensifies. We have developed a method using high-performance liquid chromatography (HPLC) equipped with a coulometric electrode array detector for separation and sensitive detection of daidzein (Da), equol (Eq), genistein (Ge) and enterolactone (Enl) in dried blood spots (DBS). Detection limits ranged from 4.5 pg or 0.09 ng/mL (Eq) to 19 pg or 0.38 ng/mL (Ge) on column. Signal linearities ranged from detection limits to 200 ng/mL (Eq, Enl) and 600 ng/mL (Da, Ge) sample concentration. Correlations between DBS and serum concentrations were 0.66 (Enl), 0.88 (Eq), 0.98 (Ge) and 0.99 (Da). Intra-assay coefficients of variation (CVs) were less than 8% and inter-assay CVs ranged from 2.4 to 20.2% for Da, Eq and Ge for three levels of controls. Enl intra-assay CV was 13.6% for the low pooled control. Analytic recovery ranged from 87% (inter-assay Ge) to 98% (inter-assay Enl). DBS concentrations of Da, Ge and Eq were stable for at least 8 weeks at 4 and 25 degrees C, and at 37 degrees C for at least 5 weeks, with Enl showing greater variability at all temperatures but relative stability for 7 weeks. Measurement of samples from 135 perimenopausal Japanese women consuming habitual diets in Kyoto and Fukushima prefectures showed the former to have the expected lower concentrations of Da and Eq (416 and 87 nM) as well as Enl (49 nM) compared to the latter locale (566, 145 and 72 nM, respectively). This method could be useful in large epidemiological research or detailed physiological studies.     

Improved clean-up procedure for the high-performance liquid chromatographic assay of clomipramine and its demethylated metabolite in human plasma

A rapid and selective assay of clomipramine and its metabolite desmethylclomipramine in human plasma, based on high-performance liquid chromatography with UV detection has been developed. The compounds were subjected to solid-phase extraction, using Extrelut 1 cartridges. Recoveries ranged between 88–95% for clomipramine, and 75–80% for desmethylclomipramine. This method has been used for therapeutic monitoring of clomipramine and its metabolite in individuals treated with this drug.     

Sensitive high-performance liquid chromatographic method for profiling phytoestrogens using coulometric electrode array detection: application to plasma analysis.

An HPLC method for profiling 13 phytoestrogens and their metabolites using coulometric electrode array detection was developed. Sensitivity of the method was slightly less than that of our GC-MS method, but significantly higher compared to the HPLC methods using diode-array or UV detection. Detection limits varied from 3.4 (secoisolariciresinol) to 40.3 (genistin) pg on column. Signal linearities ranged from the detection limits to 61 ng on column. Resolution values for the peak pairs varied from 1.1 (O-desmethylangolensin-anhydrosecoisolariciresinol) to 16 (daidzin-genistin). Intra- and interassay retention time variations were negligible and detector response variation was eliminated by frequent calibration. Chromatographic method was applied to plasma analyses and 6 of the 13 compounds were detected. Method accuracy for those six analytes varied from 69% (enterodiol) to 118% (genistein). Intraassay precision CVs ranged from 1.5% (enterolactone, 12.4 nmol/liter) to 14% (genistein, 245 nmol/liter) and interassay precision CVs ranged from 9.9% (daidzein, 67.4 nmol/liter) to 44% (enterodiol, 1.20 nmol/liter).     

Improved micro-method for the high-performance liquid chromatographic determination of caffeine and paraxanthine in biological fluids

A high-performance liquid chromatographic procedure is reported for reproducibly and sensitively quantitating caffeine and its N-demethylated metabolite paraxanthine in micro-samples. A 5-μm reversed-phase radial compression column and 214-nm fixed wavelength ultraviolet detector were used to attain a sensitivity sufficient to quantitate these compounds at concentratios as low as 80 ng/ml using only 25 μl of sample. The assay is applicable to microliter samples of whole blood, serum, plasma, saliva, amniotic, cerebro-spinal and gastric fluids such as might be obtained in studies involving small animals or neonates. The utility of the assay is illustrated with caffeine and paraxanthine levels measured in several maternal and fetal fluids following constant-rate intravenous infusion of caffeine into a rabbit throughout pregnancy.     

Determination of cyclizine and norcyclizine in plasma and urine using gas—liquid chromatography with nitrogen selective detection

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of 8-chloro-6-(2-chlorophenyl)-4H-imidazo-[1,5-a]-[1,4]-benzodiazepine-3-carboxamide [I] and its 4-hydroxy metabolite, 8-chloro-6-(2-chlorophenyl)-4-hydroxy-4H-imidazo-[1,5-a][1,4]-benzodiazepine-3-carboxamide [II] in whole blood, plasma or urine. The assay for both compounds involves extraction into diethyl ether—methylene chloride (70:30) from blood, plasma, or urine buffered to pH 9.0. The overall recoveries of [I] and [II] are 92.0 ± 5.4% (S.D.) and 90.3 ± 4.9% (S.D.), respectively. The sensitivity limit of detection is 50 ng/ml of blood, plasma, or urine using a UV detector at 254 nm. The HPLC assay was used to monitor the blood concentration—time fall-off profiles, and urinary excretion profiles in the dog following single 1 mg/kg intravenous and 5 mg/kg oral doses, and following multiple oral doses of 100 mg/kg/day of compound [I].     

Enantioselective high-performance liquid chromatography determination of methadone enantiomers and its major metabolite in human biological fluids using a new derivatized cyclodextrin-bonded phase

The simultaneous determination of methadone (Mtd) enantiomers and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in human urine and serum by enantioselective HPLC using a new Cyclobond I-2000 RSP column is described. After alkaline extraction from urine or serum with estazolam as an internal standard, Mtd enantiomers and its metabolite (EDDP) are separated on the previous column with reversed-mobile phase and detected at 210 nm. Peak resolutions are about 2.0 for Mtd enantiomers. The relative standard deviations (R.S.D.) of Mtd and EDDP standards are between 0.5 and 4.5%. Most drugs of abuse are shown not to interfere with this technique. The method has been applied to study levels of each Mtd enantiomer and of its racemic metabolite in urine and serum of patients under maintenance treatment for opiate dependence. In urine, R-(−)-Mtd levels are always higher (about 2±0.5-fold_ than those of S-(+)-Mtd and in most cases, metabolite concentrations are greater than those of global Mtd enantiomers. However, the R-(−) enantiomer levels of residual drug in serum of some patients were lower than those of its antipode. This method is suitable for pharmacokinetic and toxicological studies of Mtd enantiomers and its major metabolite in biological fluids.     

Stereoselective high-performance liquid chromatographic determination of ketamine and its active metabolite, norketamine, in human plasma

A stereoselective high-performance liquid chromatographic method for the determination of the enantiomers of ketamine and its active metabolite, norketamine, in human plasma is described. The compounds were extracted from plasma by liquid–liquid extraction three times in a combination of cyclohexane with 2.5 M NaOH, 1 mM HCl and 1 M carbonate buffer. Stereoselective separation was achieved on a Chiralcel OD column with a mobile phase of n-hexane–2-propanol (98:2, v/v). The detection wavelength was 215 nm. The lower limits of the determination of the method were 5 ng/ml for ketamine and 10 ng/ml for norketamine. The intra- and inter-day coefficients of variation ranged from 2.9 to 9.8% and from 3.4 to 10.7% for all compounds, respectively. The method was sensitive and sufficiently reproducible for stereoselective monitoring of ketamine and norketamine in human plasma during pharmacokinetic studies after the administration of ketamine for analgesia.     

Enantioselective determination of sotalol enantiomers in biological fluids using high-performance liquid chromatography

A simple and sensitive high-performance liquid chromatograhic (HPLC) method for the determination of (+)-(S)-sotalol and (−)-(R)-sotalol in biological fluids was established. Following extraction with isopropyl alcohol from biological samples on a Sep-Pak C₁₈ cartridge, the eluent was derivatized with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosol isothiocyanate (GITC). The diastereoisomeric derivatives are resolved by HPLC with UV detection at 225 nm. Calibration was linear from 0.022 to 4.41 μg/ml in human plasma and from 0.22 to 88.2 μg/ml in human urine for both (+)-(S)- and (−)-(R)-sotalol. The lower limit of determination was 0.022 μg/ml for plasma and 0.22 μg/ml for urine. The within-day and day-to-day coefficients of variation were less than 7.5% for each enantiomer at 0.09 and 1.8 μg/ml in plasma and at 0.44 and 4.4 μg/ml in urine. The method is also applicable to other biological specimens such as rat, mouse and rabbit plasma.     

High-performance liquid chromatographic determination of licochalcone A and its metabolites in biological fluids

A high-performance liquid chromatographic method has been developed for the analysis of the novel antiparasitic agent, licochalcone A (Lica), and three of its glucuronic acid conjugates in plasma and urine. The high-performance liquid chromatography assay was performed using gradient elution and UV detection at 360 nm. The proposed technique is selective, reliable and sensitive. The limits of quantification for Lica are 0.2 μg/ml in plasma and 0.14 μg/ml in urine, 1.2 μg/ml for the 4′-glucuronide in plasma and 1.4 μg/ml in urine, and 2.0 μg/ml for the 4-glucuronide in plasma and 3.2 μg/ml in urine. The reproducibility of the analytical method according to the statistical coefficients is 7% or below. The accuracy of the method is good, that is, the relative error is below 10%. The stability of Lica and its glucuronides in urine and plasma samples has been assessed during storage in the autosampler and freezer. The applicability of the assay for determining Lica and its intact glucuronide conjugates in biological fluids was shown using a single dose study in rat.     

Sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its N-desethyl metabolite in human saliva or cerebrospinal fluid using solid-phase extraction

A sensitive reversed-phase high-performance liquid chromatographic method for the determination of atevirdine and its primary metabolite in human saliva or cerebrospinal fluid using solid-phase extraction is described. Samples mixed with internal standard and sodium phosphate buffer were applied to an activated C₁₈ solid-phase extraction column. The reconstituted eluate was injected onto a Zorbax RX C₈ column utilizing a mobile phase of 100 mM ammonium acetate (pH 4.0)–isopropyl alcohol–acetonitrile (55:20:25, v/v/v). Fluorescence detection was employed with excitation at 295 nm and emission at 456 nm. Quantitation was achieved using peak-height ratios. The detection response curve was linear from 2 to 850 nM for atevirdine in both human saliva and cerebrospinal fluid and from 2 to 250 nM for the metabolite in human saliva. The method was utilized to analyze cerebrospinal fluid and saliva samples from clinical studies.     

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.