Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatography-mass spectrometry

A method is proposed for the determination of nicotine and cotinine in human urine, plasma and saliva. Nicotine and cotinine were extracted from alkalinized sample with ethyl ether and concentrated to minimum volume with nitrogen stream. The volatility of nicotine was prevented by the addition of acetic acid to the organic solvent during evaporation. Peak shapes and quantitation of nicotine and cotinine are excellent, with linear calibration curves over a wide range of 1-10,000 ng/ml. The detection limits of nicotine and cotinine are 0.2 ng/ml in urine and 1.0 ng/ml in plasma and saliva. The intra-day precision of nicotine and cotinine in all samples was <5% relative standard deviation (RSD). Urine, plasma and saliva samples of 303 non-smoking and 41 smoking volunteers from a girl's high school in Korea were quantified by the described procedure. As a result, the concentrations of nicotine and cotinine in plasma ranged from 6 to 498 ng/ml and 4 to 96 ng/ml. Otherwise, those of nicotine and cotinine in saliva ranged from 0 to 207 ng/ml and 0 to 42 ng/ml, and those of nicotine and cotinine in urine ranged from 0 to 1,590 ng/ml and 0 to 2,986 ng/ml, respectively. We found that the concentration of cotinine in plasma was successfully predicted from the salivary cotinine concentration by the equation y=2.31x+4.76 (x=the concentration of cotinine in saliva, y=the concentration of cotinine in plasma). The results show that through the accurate determination of cotinine in saliva, the risk of ETS-exposed human can be predicted.     

Effect of dietary fiber, glucomannan, on absorption of sulfonylurea in man

In order to clarify whether a dietary fiber has any effect upon the intestinal absorption of sulfonylurea, changes in plasma concentration of glibenclamide were determined during a six-hour period in nine healthy volunteers who took 2.5 mg of glibenclamide together with a breakfast and 3.9 g of glucomannan in a form of konjac powder and were compared with those of the control experiment in which the same amount of the hypoglycemic agent was given without the dietary fiber. In the control, mean plasma glibenclamide level increased rapidly, reaching a peak at 60 min and decreased gradually thereafter, whereas an increase in plasma glibenclamide level was blunted in the test experiment, thus plasma concentration of glibenclamide being lower at 30, 60, 90 and 150 min compared with the corresponding value of the control (31.7 +/- 24.5 ng/ml vs 76.4 +/- 25.0 ng/ml at 30 min; 51.3 +/- 35.5 ng/ml vs 120.9 +/- 56.0 ng/ml at 60 min; 60.0 +/- 38.8 ng/ml vs 117.4 +/- 53.1 ng/ml at 90 min; 54.0 +/- 31.5 ng/ml vs 100.7 +/- 46.5 ng/ml at 150 min). Mean plasma glucose concentration was significantly lower at 30 min in the test experiment than in the control despite the lower level of plasma glibenclamide in the former. The results suggest that glucomannan may influence the intestinal absorption of glibenclamide. A dietary fiber must be prescribed in due consideration of these facts.     

Effect of time and temperature on bovine serum and plasma progesterone concentration

Whole blood, with and without anticoagulant, from 5 pregnant cows was incubated at 40°C for 0 (30 minutes after collection), 6 and 24 hours (hr) before the blood was centrifuged and the plasma or serum was frozen for later progesterone assay. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 1.7 ng/ml at 6 hr (P < 0.01) and to 2.8 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 3.9 ng/ml at 6 hr (P < 0.01) and to 4.4 ng/ml at 24 hr (P < 0.01). Whole blood samples with and without EDTA were also incubated at 4°C for 24 hr. Mean plasma progesterone concentration decreased from 6.6 ng/ml at 0 hr to 4.2 ng/ml at 24 hr (P < 0.01). Mean serum progesterone concentration decreased from 6.1 ng/ml at 0 hr to 4.7 ng/ml at 24 hr (P < 0.01). The incubation time and temperature of whole blood, from collection of blood to the separation of serum or plasma, significantly affects assayable concentration of progesterone.     

Pulsatile nature of luteinizing hormone release is maintained during luteinizing hormone-releasing hormone stimulation in normal male rats

The plasma LH concentration is believed to be reasonably steady in normal male rats. We found that LH is released in a regular pulsatile fashion. The overall mean concentration of plasma LH in normal male rats was 46.6 +/- 4.4 (mean +/- SEM) ng/ml. The normal male rats showed periodic LH pulses: the mean pulse amplitude was 144.4 +/- 25.5 ng/ml and the inter-peak interval was 22.5 +/- 2.0 min. Each pulse lasted 9.7 +/- 0.8 min. When LH-RH (1 microgram/kg) was injected as a bolus, the peak concentration was attained in 10-30 min reaching a peak concentration of 279.4 +/- 39.6 ng/ml. Distinct pulsatile bursts of plasma LH were discernible during the period of elevated plasma LH concentration. When a higher dose of LH-RH (5 micrograms/kg) was administered, the LH concentration slowly increased to a peak concentration of 400.2 +/- 38.7 ng/ml in 20-40 min. The pulsatile nature of the LH concentration was recognizable with distinct bursts. We have observed that: (a) normal male rats release LH in a pulsatile fashion with an approximate 20-min inter-peak interval; (b) mean LH pulses last less than 10 min, and (c) the LH pulses are visible even with elevated LH and LH-RH concentrations in the general circulation.     

Occurrence of isomeric dehydrocholesterols in human plasma.

Three isomeric dehydrocholesterols were found in plasma from healthy subjects and patients with abnormal production or metabolism of cholesterol. These chemically labile steroids were isolated by a mild liquid-solid extraction procedure using octadecylsilane-bonded silica as sorbent. Sterol-protein interactions were minimized by diluting plasma with aqueous isopropanol. The dehydrocholesterols were identified by high-performance liquid chromatography-ultraviolet spectroscopy and gas chromatography-mass spectrometry as cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), 5 alpha-cholesta-6,8(9)-dien-3 beta-ol (isodehydrocholesterol), and tentatively as cholesta-5,8(9)-dien-3 beta-ol. There was a strong positive correlation between plasma levels of the two former compounds, isodehydrocholesterol levels usually being about 1.4 times higher than those of 7-dehydrocholesterol. The median concentration of 7-dehydrocholesterol in plasma from healthy subjects was 52 ng/ml. Similar concentrations were found in colectomized patients (median concentration 47 ng/ml) and patients with extrahepatic cholestasis and alcoholic liver cirrhosis (median concentrations 79 and 67 ng/ml, respectively). Patients with ileal resection or under treatment with cholestyramine had elevated levels (median concentrations 142 and 160 ng/ml, respectively) whereas patients with primary biliary cirrhosis had subnormal levels (median concentration 26 ng/ml). The results are consistent with a positive correlation between levels of the dehydrocholesterols in plasma and the rate of cholesterol synthesis. The sterols were also analyzed in human skin and bile and the results indicate that the liver may be an important source of isodehydrocholesterol.     

Determination of cabergoline and L-dopa in human plasma using liquid chromatography-tandem mass spectrometry

We determined cabergoline and L-dopa in human plasma using liquid chromatography-mass spectrometry with tandem mass spectrometry (LC-MS-MS). The deproteinized plasma samples with organic solvent or acid were analyzed directly by reversed-phase liquid chromatography. Using multiple reaction monitoring (MRM, product ions m/z 381 of m/z 452 for cabergoline and m/z 152 of m/z 198 for L-dopa) on LC-MS-MS with electrospray ionization (ESI), cabergoline and L-dopa in human plasma were determined. Calibration curves of the method showed a good linearity in the range 5-250 pg/ml for cabergoline and 1-200 ng/ml for L-dopa, respectively. The limit of determination was estimated to be approximately 2 pg/ml for cabergoline and approximately 0.1 ng/ml for L-dopa, respectively. The method was applied to the analysis of cabergoline and L-dopa in plasma samples from patients treated with these drugs. The precision of analysis showed coefficients of variation ranging from 3.8% to 10.5% at cabergoline concentration of 13.8-26.2 pg/ml and from 2.9% to 8.9% at an L-dopa concentration of 302.5-522.1 ng/ml in patient plasma. As a result, the procedure proved to be very suitable for routine analysis.     

Radioimmunoassay of [D-Trp6]-luteinizing hormone-releasing hormone: its application to animal pharmacokinetic studies after single injection and long-acting formulation administration

A sensitive radioimmunoassay (RIA) for [D-Trp6]-luteinizing hormone-releasing hormone (LHRH) has been developed. This assay allowed measurement of the LHRH analog in unextracted plasma with a minimum detectable concentration of 10 pg/ml. Validation of plasma assays was performed through Sep-Pak and HPLC purification. The in vivo fate of the peptide was investigated in dogs after subcutaneous or intravenous injections. In both cases, the LHRH analog showed longer plasma half-life than native LHRH with an elimination half-life superior to 80 min. Long-acting formulations were tested in dogs and rats: the day following administration, [D-Trp6]-LHRH plasma level rose to 2.9-4.6 ng/ml in dogs and 0.8-3.8 ng/ml in rats. From day 4 to day 30, [D-Trp6]-LHRH plasma level followed a plateau with concentrations of 0.3-0.8 ng/ml in dogs and 0.2-0.4 ng/ml in rats. In parallel, testosterone plasma concentration was reduced to castrate level between day 4 and day 7 in dogs and was significantly lowered in rats. This sensitive [D-Trp6]-LHRH RIA will be particularly useful for the evaluation of long-acting formulations in patients with advanced prostate cancer.     

Kinetic difference of berberine between hippocampus and plasma in rat after intravenous administration of Coptidis rhizoma extract

In order to investigate the pharmacokinetics of berberine in Coptidis rhizoma extract in rat hippocampus and plasma, a simple and accurate high-performance liquid chromatography method was employed in this study. Berberine was determined using a Hypersil C(18) column with an isocratic mobile phase of acetonitrile-0.05 M potassium dihydrogen phosphate (containing 0.5% triethylamine, pH 3.0) and with UV detection at 236 nm. The lower limit of quantification for berberine in both hippocampus and plasma was 24 ng/ml, and the lowest concentrations of berberine determined in rat hippocampus and plasma samples were 30.7 ng/ml at 48 h and 38.5 ng/ml at 4 h, respectively. The calibration curve for berberine was linear over the concentration range 24--6000 ng/ml. At this concentration range, the overall recoveries (90.6--94.2%) for berberine were determined and the accuracy of intra- and inter-day assays from rat samples were less than 7% RSD. Following intravenous administration of C. rhizoma extract at a dose of 10.2 mg/kg containing 3 mg/kg berberine, berberine in the plasma eliminated rapidly (t(1/2 beta)=1.13 h). However, berberine in the hippocampus increased rapidly (t(1/2 alpha)=0.215 h), peaked at 3.67 h with a concentration of 272 ng/g, and had a slow elimination rate (t(1/2 beta)=12.0 h), which suggests that berberine could have a direct action on neuron and accumulate in the hippocampus. This study first showed the pharmacokinetic characteristics of berberine in rat hippocampus and the kinetic characteristics of berberine are dissimilar in the hippocampus and plasma.     

Efficient assay for the determination of atenolol in human plasma and urine by high-performance liquid chromatography with fluorescence detection

An efficient method for the determination of atenolol in human plasma and urine was developed and validated. α-Hydroxymetoprolol, a compound with a similar polarity to atenolol, was used as the internal standard in the present high-performance liquid chromatographic analysis with fluorescence detection. The assay was validated for the concentration range of 2 to 5000 ng/ml in plasma and 1 to 20 μg.ml in urine. For both plasma and urine, the lower limit of detection was 1 ng/ml. The intra-day and inter-day variabilities for plasma samples at 40 and 900 ng/ml, and urine samples at 9.5 μg/ml were <3% (n=5).     

Platelet quantification and growth factor analysis from platelet-rich plasma: implications for wound healing

Growth factors released from activated platelets initiate and modulate wound healing in both soft and hard tissues. A recent strategy to promote the wound-healing cascade is to prepare an autologous platelet concentrate suspended in plasma, also known as platelet-rich plasma, that contains growth factors and administer it to wound sites. The purpose of this study was to quantitate platelet number and growth factors released from a prepared platelet concentrate. Whole blood was drawn from 10 healthy patients undergoing cosmetic surgery and concentrated into platelet-rich plasma. Platelet counts on whole blood and platelet-rich plasma were determined using a Cell-Dyn 3200. Platelet-derived growth factor-BB, transforming growth factor-beta1, vascular endothelial growth factor, endothelial growth factor, and insulin-like growth factor-1 were measured in the platelet-rich plasma using the enzyme-linked immunosorbent assay method. In addition, platelet activation during the concentration procedure was analyzed by measuring P selectin values in blood serum. An 8-fold increase in platelet concentration was found in the platelet-rich plasma compared with that of whole blood (baseline whole blood, 197 +/- 42 x 10 platelets/microl; platelet concentrate, 1600 +/- 330 x 10 platelets/microl). The concentration of growth factors also increased with increasing platelet number. However, growth factor concentration varied from patient to patient. On average for the whole blood as compared with platelet-rich plasma, the platelet-derived growth factor-BB concentration increased from 3.3 +/- 0.9 ng/ml to 17 +/- 8 ng/ml, transforming growth factor-beta1 concentration increased from 35 +/- 8 ng/ml to 120 +/- 42 ng/ml, vascular endothelial growth factor concentration increased from 155 +/- 110 pg/ml to 955 +/- 1030 pg/ml, and endothelial growth factor concentration increased from 129 +/- 61 pg/ml to 470 +/- 320 pg/ml. No increase was found for insulin-like growth factor-1. In addition, no increase in platelet activation occurred during the concentration procedure as determined by the platelet surface receptor P selectin (45 +/- 16 pg/ml to 52 +/- 11 pg/ml, p = 0.65). In conclusion, a variety of potentially therapeutic growth factors were detected and released from the platelets in significant levels in platelet-rich plasma preparations. Sufficient concentrates and release of these growth factors through autologous platelet gels may be capable of expediting wound healing in a variety of as yet undetermined specific wound applications.     

Feedback regulation of gonadotropic hormone secretion in neonatal pigs

The effect of castration and of administration of charcoal-treated porcine follicular fluid (pFF) containing inhibin-like activity on plasma concentration of gonadotropic hormones was studied in neonatal pigs. Plasma follicle-stimulating hormone (FSH) concentration averaged 25.1 +/- 1.5 ng/ml (mean +/- SEM) in 1-wk-old females and gradually declined to 20.2 +/- 0.7 ng/ml 6 wk later. Ovariectomy did not significantly influence plasma FSH concentration. In males, concentration averaged 8.0 +/- 0.7 ng/ml before castration but rose significantly within 2 days after castration. Injection of luteinizing hormone-releasing hormone (LHRH) did not influence plasma FSH concentrations in intact males, but did in females and in 7-wk-old males castrated at 1 wk. Plasma luteinizing hormone (LH) concentrations in 1-wk-old females (2.2 +/- 0.4 ng/ml) gradually declined and were not influenced by castration. Concentrations of plasma LH in 1-wk-old male piglets (2.8 +/- 0.7 ng/ml) were not significantly influenced by castration within 2 days but were significantly higher 6 wk later. LHRH induced a significant rise in plasma LH concentrations in all animals. Injection of pFF resulted in a decline of plasma FSH concentrations in intact and castrated males and in intact females, but did not influence plasma LH concentrations. These data demonstrate a sex-specific difference in the control of plasma FSH, but not in plasma LH concentration in the neonatal pig. Plasma FSH concentrations, but not plasma LH concentrations, are suppressed by testicular hormones in 1-wk-old piglets. Plasma FSH concentrations can be suppressed in both neonatal male and female pigs by injections of pFF.     

Development and validation of an HPLC method for the determination of spironolactone and its metabolites in paediatric plasma samples

An HPLC method has been developed and validated for the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples. The method utilises 200 microl of plasma and sample preparation involves protein precipitation followed by Solid Phase Extraction (SPE). Determination of standard curves of peak height ratio (PHR) against concentration was performed by weighted least squares linear regression using a weighting factor of 1/concentration2. The developed method was found to be linear over concentration ranges of 30-1000 ng/ml for spironolactone and 25-1000 ng/ml for 7 alpha-thiomethylspirolactone and canrenone. The lower limit of quantification for spironolactone, 7 alpha-thiomethylspirolactone and canrenone were calculated as 28, 20 and 25 ng/ml, respectively. The method was shown to be applicable to the determination of spironolactone, 7 alpha-thiomethylspirolactone and canrenone in paediatric plasma samples and also plasma from healthy human volunteers.     

Study of plasma aldosterone in normal pregnancy, in pre-eclamptic women and in cord plasma of newborns.

A radioimmunoassay without chromatography was used for the determination of plasma aldosterone in pregnancy. The mean values (+/- S.D.) of aldosterone concentration increased consistently from 23.2 +/- 5.3 ng/100 ml (n = 14) during the first trimester to 37.2 +/- 10.6 ng/100 ml (n = 17) during the second trimester and 64.0 +/- 18.8 ng/100 ml (n = 29) during the third trimester of pregnancy. The highest values were found at delivery (71.9 +/- 14.2 ng/100 ml; n = 21) and in the cord plasma of newborns (83.4 +/- 14.9 ng/100 ml; n = 21). Significantly lower plasma aldosterone values were found in the plasma of pre-eclamptic women during the third trimester of pregnancy (41.9 +/- 21.3 ng/100 ml; n = 11).     

Capillary gas chromatographic determination of permethrin insecticide by transesterification

A sensitive method was developed to determine permethrin extracted from phosphate buffer and cattle plasma by potassium cyanide catalyzed transesterification of this insecticide with refluxing ethanol and detection of the resulting ethyl esters by capillary gas chromatography with an electron capture detector. With a reflux time of 2 h and with 3-phenoxybenzyl 2-chlorobenzoate as an internal standard, linear calibration curves from buffer (5–250 ng) and plasma (5–100 ng) were obtained. Precision and accuracy of the method were 15%. The limit of detection was approximately 2.5 ng/ml (cis) and 1 ng/ml (trans) from buffer. In cattle sprayed along the back at 2 mg/kg, the concentration of cis- and trans-permethrin in plasma was below the detection limit (5 ng/ml).     

Selective determination of sultopride in human plasma using high-performance liquid chromatography with ultraviolet detection and particle beam mass spectrometry

We developed a sensitive and selective method for determining levels of sultopride, a neuroleptic drug of the substituted benzamide, in human plasma using high-performance liquid chromatography (HPLC) combined with UV detection and particle beam mass spectrometry (PBMS). Sutopride was extracted with tert.-butylmethyl ether using a salting-out technique. Tiapride served as an internal standard (I.S.). Sutopride and I.S. were separated by HPLC on a silica column with a mobile phase of acetonitrile-0.1 M ammonium acetate (94:6, v/v). The calibration curves were linear over the concentration range from 5 to 1000 ng/ml by HPLC with UV detection and from 10 to 1000 ng/ml with PBMS detection. The limit of quantitation was 5 ng/ml with UV detection and 10 ng/ml with PBMS detection. The absolute recovery was 92% and the within-day coefficients of variation were 2.9–7.1% at plasma concentrations from 50 to 500 ng/ml, determined by HPLC with UV detection. Using this method, we measured the plasma concentrations of sultopride with replicate analyses in four hospitalized patients and steady-state plasma levels were determined to be 161.6±30.8, 321.1±93.7, 726.5±143.1 and 1273.6±211.2 ng/ml, respectively.     

Development of a high-performance liquid chromatographic method to determine the concentration of karenitecin,a novel highly lipophilic camptothecin derivative,in human plasma and urine

Karenitecin is a novel, highly lipophilic camptothecin derivative with potent anticancer potential. We have developed a sensitive high-performance liquid chromatographic method for the determination of karenitecin concentration in human plasma and urine. Karenitecin was isolated from human plasma and urine using solid-phase extraction. Separation was achieved by gradient elution, using a water and acetonitrile mobile phase, on an ODS analytical column. Karenitecin was detected using fluorescence detection at excitation and emission wavelengths of 370 and 490 nm, respectively. Retention time for karenitecin was 16.2±0.5 min and 8.0±0.2 min for camptothecin, the internal standard. The karenitecin peak was baseline resolved, with the nearest peak at 3.1 min distance. Using normal volunteer plasma and urine from multiple individuals, as well as samples from the 50 patients analyzed to date, no interfering peaks were detected. Inter- and intra-day coefficients of variance were <4.4 and 7.1% for plasma and <4.9 and 11.6% for urine. Assay precision, based on an extracted karenitecin standard plasma sample of 2.5 ng/ml, was +4.46% with a mean accuracy of 92.4%. For extracted karenitecin standard urine samples of 2.5 ng/ml assay precision was +2.35% with a mean accuracy of 99.5%. The mean recovery of karenitecin, at plasma concentrations of 1.0 and 50 ng/ml, was 81.9 and 87.8% respectively. In urine, at concentrations of 1.5 and 50 ng/ml, the mean recoveries were 90.3 and 78.4% respectively. The lower limit of detection (LLD) for karenitecin was 0.5 ng/ml in plasma and 1.0 ng/ml in urine. The lower limit of quantification (LLQ) for karenitecin was 1 ng/ml and 1.5 ng/ml for plasma and urine, respectively. Stability studies indicate that when frozen at −70°C, karenitecin is stable in human plasma for up to 3 months and in human urine for up to 1 month. This method is useful for the quantification of karenitecin in plasma and urine samples for clinical pharmacology studies in patients receiving this agent in clinical trials.     

Simultaneous determination of tramadol and its major active metabolite O-demethyltramadol by high-performance liquid chromatography with electrochemical detection

A novel, highly sensitive method was developed for simultaneous determination of tramadol and its main active metabolite O-demethyltramadol (ODMT) in rat plasma. The method involves a single-step extraction procedure and a specific determination by high-performance liquid chromatography with electrochemical detection, using an ethoxy analogue of tramadol (L-233) as internal standard. The dual-electrode detector was operated in the oxidation-screening mode. Absolute recoveries of tramadol and ODMT were about 80%. Calibration curves were linear over a concentration range of 10–1000 ng/ml for ODMT and 10–10 000 ng/ml for tramadol with intra- and inter-day coefficients of variation not exceeding 10% and 15%, respectively. The limit of quantification for tramadol and ODMT was lower than 15 ng/ml and 10 ng/ml using 100 μl of plasma, respectively. The described method allows an adequate characterization of the plasma vs. time profiles for both compounds.     

Levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma reflect rates of bile acid synthesis in man

A method for analysis of 7 alpha-hydroxy-4-cholesten-3-one in plasma is described. Following solid-phase extraction/purification the compound is determined by high-performance liquid chromatography using a UV detector. The median concentration in healthy subjects was 12 ng/ml (range 3-40). The levels were lower in diseases associated with a low bile acid production: extrahepatic cholestasis, less than 1.5 ng/ml (range less than 0.9-3); liver cirrhosis less than 1.5 ng/ml (range less than 0.9-38), and higher in diseases associated with a high bile acid production: cholestyramine treatment, 188 ng/ml (range 54-477); ileal resection 397 ng/ml (range 128-750). The levels were essentially normal in patients with colon resection. The results are consistent with a strong positive correlation between the levels of 7 alpha-hydroxy-4-cholesten-3-one in plasma and the rate of bile acid synthesis.     

Size heterogeneity of epidermal growth factor in human body fluids

We measured the concentration of immunoreactive (IR) hEGF in various body fluids by radioimmunoassay (RIA) and evaluated its size heterogeneity by size exclusion high performance liquid chromatography combined with RIA or with time-resolved immunoflurometric assay (TR-IFMA). Mean concentration was 80 ng/ml in urine, 65 ng/ml in milk, 50 ng/ml in seminal plasma, 25 ng/ml in armpit sweat, 1 ng/ml in breast sweat, 0.3 ng/ml in third-trimester amniotic fluid, 3 ng/ml in saliva, 1.5 ng/ml in tears and 0.3 ng/ml in gastric juice.

All the fluids except armpit sweat and gastric juice contained two to five molecular sizes of IR-hEGF. As well as the 6200-dalton (6.2kDa) hEGF we found at least four other different molecular sizes with approximate weights of 300, 150, 70 and 20kDa. The authentic 6.2kDa form made up >90% of the total IR-hEGF in all except the amniotic fluid where its proportion was 71%, and the seminal plasma where the proportion could not be determined.     

Determination of benflumetol in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

A reversed-phase high-performance liquid chromatographic method for the determination of benflumetol in human plasma is described. Benflumetol in plasma samples was extracted with a glacial acetic acid-ethyl acetate (1:100, v/v) mixture at pH 4.0. Chromatography was performed on a Spherisorb C₁₈ column using a methanol-water-glacial acetic acid-diethyl amine (93:6:1:0.03, v/v) mixture as the mobile phase and UV-VIS detection at 335 nm. The identity and purity of the benflumetol peak were carefully examined, and the internal standard method was applied for its quantitation. The absolute recovery of benflumetol in spiked plasma samples was 92.91% over the concentration range 5–4000 ng/ml. The recovery of internal standard “8212” at a concentration of 300 ng/ml in spiked plasma was 84.85%. The detection limit of benflumetol was 11.8 ng/ml. Plasma concentration-time profiles in healthy volunteer adults were measured after a single-dose oral administration of 500 mg of benflumetol. The assay procedures were within the quality control limits.