Determination of Org NC 45 (a myoneural blocking agent) in human plasma using high-performance normal-phase liquid chromatography

The assay for the quantification of Org NC 45 in human plasma is described in detail. It comprises ion-pair extraction and normal-phase liquid chromatography in conjunction with UV detection. An analogue, 17β-deacetyl-Org NC 45, is used as standard in the assay procedure. The accuracy and precision of the assay at 400 ng of Org NC 45 per ml of plasma are 0.7% and 2.8%, respectively. The detection limit is approx. 50 ng Org NC 45 per ml of plasma. The assay can be used for the pharmacokinetic evaluation of Org NC 45 in man.     

Sensitive method for the simultaneous measurement of fluphenazine decanoate and fluphenazine in plasma by high-performance liquid chromatography with cuolometric detection

A highly sensitive and specific high-performance liquid chromatographic method with coulometric detection was developed for the simultaneous assay of fluphenazine decanoate and fluphenazine in plasma. The extraction and sample clean-up procedures are simple, rapid to execute, yet yield chromatograms relatively free of any interference from endogenous plasma constituents, such that the extraordinary sensitivity of the coulometric detector can be exploited fully. This is the first analytical procedure for the simultaneous determination of fluphenazine decanoate and fluphenazine. The detection limits for both fluphenazine decanoate and fluphenazine were 0.1 ng/ml plasma and the limits of quantitation were 0.25 ng/ml plasma. Standard curves from 0.25 to 10 ng/ml were linear with coefficients of variation <10%. The method was applied to measure plasma levels of fluphenazine decanoate and fluphenazine in patients under medication with 25–50 mg biweekly intramuscular (i.m.) injections of fluphenazine decanoate. It was possible to monitor the plasma levels of fluphenazine in all cases. Fluphenazine decanoate was present in measurable concentration in the plasma of 4 out of 5 patients who received biweekly i.m. injections of 50 mg fluphenazine decanoate. In a pilot experiment with a dog, the method was used to follow fluphenazine decanoate and fluphenazine plasma levels up to 13 days, at least, after i.m. single dose (10 mg/kg).     

Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 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.     

Simultaneous determination of tanshinone IIA and cryptotanshinone in rat plasma by liquid chromatography-electrospray ionisation-mass spectrometry

Tanshinone IIA (TS) and cryptotanshinone (CT) are the major active constituents contained in Radix salvia miltiorrhiza. This paper described a rapid, sensitive and specific assay for the simultaneous quantitative determination of TS and CT in rat plasma. After a single step of liquid-liquid extraction, plasma samples were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) using a reversed-phase C18 column (150 mmx2.0 mm, 5 microm, Shim-pack VP-ODS column). The assay was linear in the concentration range of 2-200 ng/ml. The lower limits of quantification of TS and CT were 1 and 0.2 ng/ml, respectively. Recoveries of TS and CT were greater than 80%. The precisions and accuracies determined from 5 days were all within 12%. The assay was applied to a pharmacokinetic study in rats after an oral administration of total tanshinones with a dose of 150 mg/kg (containing 12% of TS and CT). Results showed that this simple and rapid method was sensitive enough to follow the plasma levels of TS and CT in rats, even though the concentration maximums of both were below 20 ng/ml after an oral administration of total tanshinones.     

Analysis of 6-mercaptopurine in human plasma with a high-performance liquid chromatographic method including post-column derivatization and fluorimetric detection

A relatively simple assay with improved reliability and sensitivity for measuring levels of 6-mercaptopurine in human plasma is presented. After extraction of the compound and the added internal standard with phenyl mercury acetate, samples were separated by ion-pair reversed-phase high-performance liquid chromatography. On-line the analytes were oxidized to fluorescent products and detected in a flow-fluorimeter. The within-day coefficient of variation was 3.8% at a concentration of 25 ng/ml. The lower detection limit was 2 ng/ml when 1.0 ml of plasma was used. Mercaptopurine concentration versus time curves of two subjects after a single oral dose of azathioprine are shown.     

Determination of eperisone in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric method was developed to determine eperisone hydrochloride, 4′-ethyl-2-methyl-3-piperidinopropiophenone hydrochloride, in human plasma over the concentration range 0.2–40 ng/ml. Excellent sensitivity was achieved by selection of a favorable fragment ion, m/z 98, of eperisone and reduction of heat decomposition of eperisone by using a splitless injector and a shortened capillary column. The method described here allows the determination of plasma concentrations as low as 0.2 ng/ml, the concentration attained 6 h after a single oral administration of 50 mg. At eperisone hydrochloride concentrations higher than 0.5 ng/ml, the mean inter-day variation of accuracy of the assay was less than 12%.     

Stereospecific high-performance liquid chromatographic analysis of tramadol and its O-demethylated (M1) and N,O-demethylated (M5) metabolites in human plasma

A stereospecific method for simultaneous quantitation of the enantiomers of tramadol (T) and its active metabolites O-demethyl tramadol (M1) and O-demethyl-N-demethyl tramadol (M5) in human plasma is reported. After the addition of penbutolol (IS), plasma (0.5 ml) samples were extracted into methyl tert-butyl ether, followed by back extraction into an acidic solution. The separation was achieved using a Chiralpak AD column with a mobile phase of hexanes:ethanol:diethylamine (94:6:0.2) and a flow rate of 1 ml/min. The fluorescence of analytes was then detected at excitation and emission wavelengths of 275 and 300 nm, respectively. All the six enantiomeric peaks of interest plus three unknown metabolite peaks and IS peak (a total of 10 peaks) eluted within 23 min, free from endogenous interference. The assay was validated in the plasma concentration range of 2.5-250 ng/ml, with a lower limit of quantitation of 2.5 ng/ml, for all the six analytes. The extraction efficiency (n=5) was close to 100% for both T and M1 enantiomers and 85% for M5 and IS enantiomers. The application of the assay was demonstrated by simultaneous measurement of plasma concentrations of T, M1, and M5 enantiomers in a healthy volunteer after the administration of 50 mg oral doses of racemic T.     

Determination of telmisartan in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive method for the determination of the angiotensin II receptor antagonist, telmisartan, in human plasma has been developed. Telmisartan and the internal standard, diphenhydramine, were extracted from plasma using diethyl ether-dichloromethane (60:40, v/v), and separated on a Zorbax extend C(18) column using methanol-10mM ammonium acetate (85:15, v/v) adjusted to pH 4.5 after mixing with formic acid as mobile phase. Detection was carried out by multiple reaction monitoring on a Q-trap LC-MS/MS system with an ESI interface. The assay was linear over the range 0.5-600.0 ng/ml with a limit of quantitation of 0.5 ng/ml and a limit of detection of 0.05 ng/ml. Intra- and inter-day precision were <6.7% and <8.1%, respectively, and the accuracy was in the range 88.9-111.0%. The assay was applied to a pharmacokinetic study of telmisartan given as a single oral dose (80 mg) to healthy volunteers.     

Intranasal activity of the growth hormone releasing peptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 in conscious dogs

This series of experiments was conducted to evaluate the growth hormone (GH) releasing activity of intranasally administered His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6, SK&F 110679) in conscious dogs. Intranasal administration of GHRP-6 increased plasma growth hormone levels in the conscious dog in a dose-related manner. Doses of 0.25 and 0.5 mg/kg produced GH levels of 11.3 +/- 4.8 ng/ml and 28.6 +/- 8.0 ng/ml, respectively. Peak levels were observed 15 minutes after dosing and GH levels were elevated for up to 105 minutes after intranasal dosing. Intranasal administration of isotonic saline did not produce any change in basal (negligable) GH levels. When GHRP-6 was given by the intravenous route, a maximal dose of 0.5 mg/kg, produced a peak plasma GH concentration of 60.8 +/- 10.5 ng/ml. Saline had no effect on GH levels when given intravenously. Using the intravenous and intranasal GH response data (i.e., area under the time-response curves), the intranasal bioavailability of GHRP-6 was estimated to be 34.4 to 44.9%. The results of these studies suggest that significant activity and excellent bioavailability can be achieved when GHRP-6 is administered by the intranasal route to conscious dogs. Based on these results, the intranasal activity of GHRP-6 should be evaluated in man. The successful intranasal administration of this peptide in man should provide GH therapy with reduced patient discomfort and better patient compliance when compared to presently available parenterally administered remedies.     

High-performance liquid chromatographic assay for the measurement of the novel microtubule inhibitor 1069C85 in biological tissues and fluids

1069C85 is a novel tubulin binder developed to circumvent the resistance associated with the Vinca alkaloids. Cytotoxic activity has been demonstrated in vitro against a variety of tumour cell lines, including a variant of the P388 leukaemia with acquired resistance to vincristine. A phase I clinical trial is planned and an assay suitable for preclinical and clinical pharmacokinetics has been developed. A high-performance liquid chromatographic (HPLC) assay is described which allows measurement of 1069C85 in plasma, urine, and tissue samples. The method uses reversed-phase chromatography with isocratic elution and detection by fluorescence at 406 nm following excitation at 340 nm. The assay is specific, sensitive (limit of sensitivity 0.25 ng/ml) and reproducible (coefficient of variation <5%). The method has been used to study the pharmacokinetics of 1069C85 in Balb C mice following a single oral dose of 1 mg/kg. The maximum plasma concentration was reached 15 min after administration and subsequent elimination was slow with a half life of 6.5 ± 2.2 h. The drug remained detectable in plasma, at 1 ± 0.5 ng/ml, 24 h after this dose. This assay will be used to determine the pharmacokinetic profile of 1069C85 in mice and in a forthcoming phase I clinical trial.     

Liquid chromatography-electrospray tandem mass spectrometric assay suitable for quantitation of halofuginone in plasma

An LC-MS/MS method was developed to quantitate the potential antitumor agent halofuginone in plasma. The assay uses 0.2 ml of plasma; chlorohalofuginone internal standard; acetonitrile for protein precipitation; a Phenomenex SYNERGI 4 micro Polar RP 80A (4 microm, 100 mm x 2 mm) column; an isocratic mobile phase of methanol:water:formic acid (80:20:0.02, v/v/v); and positive-ion electrospray ionization with selective reaction monitoring detection. Halofuginone eluted at approximately 2.4 min, internal standard eluted at approximately 2.9 min, and no endogenous materials interfered with their measurement. The assay was accurate, precise, and linear between 0.1 and 100 ng/ml. Halofuginone could be quantitated in dog plasma for at least 24 h after an i.v. dose of 0.1mg/kg. The assay is being used in ongoing pharmacokinetic studies of halofuginone.     

Simultaneous determination of chloroquine and metronidazole in human biological fluid by high pressure liquid chromatography

Chloroquine (CQ) and metronidazole (MZ) were measured in human urine and plasma by HPLC with UV detection. This method was used to analyse plasma levels in 4 African volunteers after an oral dose of 1000 mg CQ and 750 mg MZ, in a European on weekly prophylaxis of 500 mg CQ, and on 50 hospital urine samples. In the Africans peak plasma levels were over 1 microgram/ml and peak time was 1 1/2-2 hr. In the European plasma levels ranged from 0.58 to 0.36 microgram/ml. Over 80% of the urine samples contained CQ, MZ or both. The assay system was found flexible and economical for the therapeutic monitoring of these two important tropical drugs.     

Apparent resistance to hypotensive effect of clonidine.

Clonidine failed to reduce the blood pressures of two patients with essential hypertension. On was given 5-4 mg/day and the other 6 mg/day, and their respective peak plasma clonidine concentrations were 26-2 ng/ml and 14-4 ng/ml. Several months after the end of clonidine treatment a single oral dose of 0-3 mg of clonidine produced maximum falls in blood pressure of 30/22 mm Hg and 88/41 mm Hg with peak plasma clonidine concentrations of 1-4 ng/ml and 0-9 ng/ml. Resistance to the hypotensive effect of high doses of clonidine may be due to stimulation of peripheral alpha-adrenoceptors causing vasoconstriction, which maintains a raised blood pressure.     

Opioid action on luteinizing hormone secretion in newborn pigs: paradoxical effect of naloxone

German Landrace piglets, 6-7 days of age, received either saline (9 males, 8 females), 0.5 mg naloxone/kg body weight (7 males, 7 females), 2.0 mg naloxone/kg (7 males, 8 females) or 0.5 mg DADLE (potent leu-enkephalin analog)/kg (7 males, 7 females) through a catheter inserted into the jugular vein 2-4 days previously. Male or female piglets were allocated randomly, within litter, to the different experimental groups. Blood samples were withdrawn for a period of 240 min at 10-min intervals for the first 60 min following injection and at 20-min intervals for the rest of the test period. Piglets were separated from their mother via a detachable wall and were allowed to suckle every 50 min. DADLE failed to alter plasma levels of LH in both males and females. Naloxone induced a significant (P less than 0.01) decrease in LH concentrations in females 10 to 60 min after injection (saline: 2.3 +/- 0.2 ng/ml plasma (SEM); 0.5 mg naloxone/kg: 1.0 +/- 0.2 ng/ml plasma and 2 mg naloxone/kg 1.2 +/- 0.4 ng/ml plasma). In males low doses of naloxone reduced plasma LH levels 10 to 40 min after injection (saline: 2.0 +/- 0.3 ng/ml plasma and 0.5 ng naloxone/kg: 1.1 +/- 0.3 ng/ml), whereas a decrease in plasma LH levels occurred 80 to 140 min after injection of high doses of naloxone (saline: 2.1 +/- 0.2 ng/ml and 2 mg naloxone/kg: 1.0 +/- 0.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of a potent LHRH antagonist (Org 30850) in female and male rats.

Org 30850 (Ac-D-pClPhe1,2,D-Bal3,D-Lys6,D-Ala10-LHRH) is a novel LHRH antagonist, which is being developed for the treatment of hormone-dependent disorders. The activities of this compound with respect to its endocrinological properties and side-effects were tested in rats and the results were compared with one of the first LHRH antagonists: Ac-D-pClPhe1,2,D-Trp3,D-Arg6,D-Ala10-LHRH (Org 30276). A single subcutaneous (s.c.) dose of 0.3 micrograms/kg Org 30850 administered to rats in pro-estrus gave inhibition of ovulation in approx. 50% of the rats, whereas Org 30276 was approx. 4 times less potent. The effect of a single s.c. injection of Org 30850 on testosterone levels in young adult male rats was also studied. The administration of 250 micrograms/kg or higher of Org 30850 induced a significant decrease in testosterone levels after 3 h, this effect lasted for at least 48 h. Treatment of female rats for 14 days with a daily dose of 12 micrograms/kg Org 30850 decreased statistically significantly uterine and ovarian weights. At a daily dose of 50 micrograms/kg Org 30850 completely suppressed estrous cycles and significantly decreased estradiol and FSH serum levels. The LH levels were below the detection level in both control and treated animals on the (expected) second day of di-estrus. Treatment of male rats for 14 days (25-200 micrograms/kg) resulted in a dose-dependent reduction of the gonads, accessory sex organs, testosterone levels and gonadotrophins. The decrease in gonadal function in both sexes was reversible since the females proved to be as fertile as the controls 6 weeks after the last treatment and an almost complete recovery of the weight of testes, seminal vesicles and ventral prostate was observed in the males 4 weeks after cessation of treatment. In contrast to Org 30276, Org 30850 exerted very slight irritation at the site of injection and no edematous reactions in the extremities at a daily dose of up to 8 mg/kg in male rats. It is concluded that Org 30850 is a very potent LHRH antagonist without edematous reactions and with a more favourable therapeutic index than Org 30276.     

Quantitation of 6-mercaptopurine in biologic fluids using high-performance liquid chromatography: A selective and novel procedure

A simple, selective, sensitive and rapid procedure is described for the quantitation of 6-mercaptopurine (6-MP) in biological fluids. A sensitivity of at least 5 ng/ml is easily achieved in plasma on a reversed-phase octadecylsilane (C₁) column using a high-performance liquid chromatography system following an initial protein precipitation and a clean-up step. Mean extractability of the drug from plasma following this procedure is greater than 98% and the overall coefficient of variation for the assay is below 6%. Plasma levels of 6-MP were measured in a rhesus monkey for 12 h following an intravenous administration of a single bolus dose (4 mg/kg) of 6-MP.     

Determination of plasma concentrations of epirubicin and its metabolites by high-performance liquid chromatography during a 96-h infusion in cancer chemotherapy

In order to determine epirubicin and its metabolites at low concentrations (<38 ng/ml) in small plasma samples, a fast reliable method based on a precipitation pre-treatment and sensitive reversed-phase isocratic HPLC has been developed and validated for epirubicin in the range 5–100 ng/ml. The R.S.D. was 5–9% over this concentration range. For human serum containing 25 ng/ml of epirubicin, the inter- and intra-day variation was <10%. Recoveries of the metabolites epirubicinol, 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone at 20 ng/ml ranged from 94–104%. The assay has been used to study human plasma samples taken during a 96-h infusion of epirubicin in a patient with multiple myeloma. The combined levels of the unseparated metabolites, epirubicin glucuronide and epirubicinol glucuronide, were semiquantitatively determined after treatment with β-glucuronidase. The metabolites epirubicinol and 7-deoxydoxorubicinolone, but not 7-deoxydoxorubicinone, were also detected and measured.     

Determination of tegaserod by LC-ESI-MS/MS and its application to a pharmacokinetic study in healthy Chinese volunteers

A simple, rapid and sensitive high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) assay for determination of tegaserod in human plasma using diazepam as internal standard (IS) was established. After adjustment to a basic pH with sodium hydroxide, plasma was extracted by ethyl acetate and separated by high performance liquid chromatography (HPLC) on a reversed-phase C18 column with a mobile phase of methanol: 5 mM ammonium acetate (75:25, v/v, adjusting the pH to 3.5 with glacial acetic acid). The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions; m/z 302.5, 173.2 and 285.4, 193.2 were measured in positive mode for tegaserod and internal standard (diazepam), respectively. The lower limit of quantification (LLOQ) was 0.05 ng/ml. The calibration curves were linear over the range 0.05-8.0 ng/ml (r=0.9996) for tegaserod. The mean absolute recovery of tegaserod was more than 85.56%. Intra- and inter-day variability values were less than 9.21% and 10.02%, respectively. The samples were stable for 8h under room temperature (25 degrees C, three freeze-thaw cycles in 30 days and for 30 days under -70 degrees C). After administration of a single dose of tegaserod maleate 4 mg, 6 mg and 12 mg, respectively, the area under the plasma concentration versus time curve from time 0 h to 12 h (AUC0-12) were (2.89+/-0.88), (5.32+/-1.21) and (9.38+/-3.42) ng h/ml, respectively; peak plasma concentration (Cmax) were (1.25+/-0.53), (2.21+/-0.52) and (4.34+/-1.66) ng/ml, respectively; apparent volume of distribution (Vd/F) were (6630.5+/-2057.8), (7615.2+/-2242.8) and (7163.7+/-2057.2) l, respectively; clearance rate (CL/F) were (1851.4+/-496.9), (1596.2+/-378.5) and (1894.2+/-459.3) l/h, respectively; time to Cmax (Tmax) were (1.00+/-0.21), (1.05+/-0.28) and (1.04+/-0.16) h, respectively; and elimination half-life (t1/2) were (3.11+/-0.78), (3.93+/-0.92) and (3.47+/-0.53) h, respectively; MRT were (3.74+/-0.85), (4.04+/-0.56) and (3.28+/-0.66) h, respectively. The essential pharmacokinetic parameters after oral multiple doses (6mg, b.i.d) were as follows: Cssmax, (2.72+/-0.61) ng/ml; Tmax, (1.10+/-0.25) h; Cssmin, (0.085+/-0.01) ng/ml; Cav, (0.54+/-0.12) ng/ml; DF, (4.84+/-0.86); AUCss, (6.53+/-1.5) ngh/ml. This developed and validated assay method had been successfully applied to a pharmacokinetic study after oral administration of tegaserod maleate in healthy Chinese volunteers at a single dose of 4 mg, 6 mg and 12 mg, respectively. The pharmacokinetic parameters can provide some information for clinical medication.     

Chiral determination of mirtazapine in human blood plasma by high-performance liquid chromatography

A method is described for the determination of the two enantiomers of mirtazapine in human blood plasma by high-performance liquid chromatography. Measurements were performed on drug free plasma spiked with mirtazapine and used to prepare and validate standard curves. Levels of enantiomers of mirtazapine were also measured in patients being treated for depression with racemic mirtazapine. Mirtazapine was separated from plasma by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved using a Chiralpak AD column and pre-column and compounds were detected by their absorption at 290 nm. Imipramine was used as an internal standard. The assay was validated for each analyte in the concentration range 10–100 ng/ml. The coefficient of variance was 16% and 5.5% for(+)-mirtazapine for 10 and 100 ng/ml control specimens respectively and 15% and 7.3% for mirtazapine for 10 and 100 ng/ml control specimens respectively. This assay is appropriate for use in the clinical range. The range of plasma mirtazapine concentrations from eleven patients taking daily doses of 30–45 mg of racemate was <5 to 69 ng/ml for (+)-mirtazapine and 13–88 ng/ml for (−)-mirtazapine for blood specimens collected 10–17.5 h after taking the dose.