Quantification of a positive inotropic agent in plasma: Towards total automation

Summary An assay for the measurement of SK&F 95654, a new positive inotropic agent in plasma is described. The assay is totally automated, is easy to use and involves minimal human input. It combines solid-phase extraction with reverse phase HPLC and UV detection at 300nm, by the use of a small robotic unit and an AASP LC module. The assay is sufficiently sensitive to measure concentrations of 0.025mg I⁻¹ in dog, rat and mouse plasma, and capable of analysing at least one hundred samples per day. The assay is robust, accurate and precise. The strategy described is widely applicable.     

Simple methodology for the therapeutic drug monitoring of the tyrosine kinase inhibitors dasatinib and imatinib

A simple HPLC method has been developed to measure imatinib and N‐desmethylimatinib (norimatinib) in plasma or serum at concentrations attained during therapy. Adaptation of this method to LC‐MS/MS also allows dasatinib assay. A small sample volume (100 μL HPLC‐UV, 50 μL LC‐MS/MS) is required and analysis time is <5 min in each case. Detection was by UV (270 nm) or selective reaction monitoring (two transitions per analyte) tandem mass spectrometry. Assay calibration was linear (0.05–10 mg/L imatinib, 0.01–2.0 mg/L norimatinib and 1–200 µg/L dasatinib), with acceptable accuracy (86–114%) and precision (<14% RSD) for both methods. A comparison between whole blood and plasma confirmed that plasma is the preferred sample for imatinib and norimatinib assay. For dasatinib, although whole blood concentrations were slightly higher, plasma is still the preferred sample. Despite considerable variation in the (median, range) plasma imatinib and norimatinib concentrations in patient samples [1.66 (0.02–4.96) and 0.32 (0.01–0.99) mg/L, respectively, N = 104], plasma imatinib was >1 mg/L (suggested target for response) in all but one sample from patients achieving complete molecular response. As to dasatinib, the median (range) plasma dasatinib concentration was 13 (2‐143) µg/L (N = 33). More observations are needed to properly assess the potential role of therapeutic drug monitoring in guiding treatment with dasatinib. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid screening for monensin residues in poultry plasma by a dry reagent dissociation enhanced lanthanide fluoroimmunoassay.

Monensin, a carboxylic acid ionophore, is commonly fed to poultry to control coccidiosis. A method for rapid analysis of unextracted poultry plasma samples has been developed based on a novel immunoassay format: one-step all-in-one dry reagent time resolved fluorimetry. All assay specific components were pre-dried onto microtitration plate wells. Only addition of the serum sample diluted in assay buffer was required to perform analysis. Results were available one hour after sample addition. The limit of detection (mean +/- 3s) of the assay calculated from the analysis of 23 known negative samples was 14.2 ng ml-1. Intra- and inter-assay RSD were determined as 15.2 and 7.4%, respectively, using a plasma sample fortified with 50 mg ml-1 monensin. Eight broiler chickens were fed monensin at a dose rate of 120 mg kg-1 feed for one week, blood sampled then slaughtered without drug withdrawal. Plasma monensin concentrations, as determined by the fluoroimmunoassay ranged from 101-297 ng ml-1. This compared with monensin liver concentrations, determined by LC-MS, which ranged from 13-41 ng g-1. The fluoroimmunoassay described is extremely user friendly, gives particularly rapid results and is suitable for the detection and quantification of plasma monensin residues. Data from medicated poultry suggest that analysis of plasma may be useful in predicting the extent of monensin liver residues.     

Determination of total platinum in plasma and plasma ultrafiltrate,from subjects dosed with the platinum-containing N-(2-hydroxypropyl)methacrylamide copolymer AP5280, by use of graphite-furnace Zeeman atomic-absorption spectrometry

AP5280 is an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer to which are linked tetrapeptide side chains containing bioactive platinum complexes at their C-terminal sides. We have developed and validated a rapid and sensitive analytical assay for the determination of total platinum concentrations in plasma, and free platinum of an AP5280 origin in plasma ultrafiltrate (PUF), of subjects dosed with AP5280. The total platinum levels were determined by use of graphite-furnace atomic-absorption spectrometry (GF-AAS) with Zeeman correction after appropriate dilution of the plasma sample with plasma-hydrochloric acid 0.2 mol L(-1) (1:5) as diluent. The limit of quantitation of this assay is 0.25 micromol L(-1) platinum in plasma. Linear calibration curves were obtained over the concentration range 0.25-5.0 micromol L(-1). Accuracy was between 87.7% and 104.2% and precision was 15.3% at the lowest concentration and less than 14% for all other levels tested. Accuracy and precision were thus in accordance with generally accepted criteria for analytical methods. Analysis of samples obtained from patients receiving AP5280 demonstrated the applicability of the described assay.Analysis of free platinum in PUF was performed by use of a previously validated and reported assay from our institute in which the same instrumental method is used.     

Sensitive high-performance liquid chromatographic assay for the determination of eugenol in body fluids

The high-performance liquid chromatographic assay described permitted a simple, rapid, sensitive, selective and precise quantitative determination of eugenol in body fluids (serum, urine and bile) without derivatization. Amounts in the range 0.02-100 micrograms of eugenol per millilitre of body fluid were determined with intra-assay coefficients of variation below 4% (3.72-1.13%). The short analysis time for each sample and the selectivity even at low concentrations made this assay suitable for pharmacokinetic studies. Eugenol undergoes a pronounced first-pass effect; in serum, unconjugated eugenol was not detected after an oral dose of 150 mg. The kinetics of eugenol conjugates were measured. More than 80% of the dose was excreted within 6 h after oral administration.     

Stereospecific high-performance liquid chromatographic determination of tocainide

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.     

High-performance liquid chromatographic determination of plasma and urinary 1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3,7-dicarboxylic acid.

A high-performance liquid chromatographic method for the analysis of 1-ethyl-1,4-dihydro-4-oxo-1,8-naphthyridine-3,7-dicarboxylic acid (I) in plasma and urine is described. A statistical evaluation of the assay technique has shown acceptable accuracy and precision at concentrations as high as 2.0 microgram/ml of plasma or 29.0 microgram/ml of urine for samples augmented with 1. As little as 0.08 microgram/ml of I in plasma or 0.42 microgram/ml of I in urine were quantitatively determined. The mean relative error for the assay of unknown concentrations of I in plasma and urine was +/- 8% and +/- 3%, respectively. This method was used for the analysis of I in the plasma and urine of rhesus monkeys following oral administration of 200 mg/kg of nalidixic acid.     

Determination by high-performance liquid chromatography of hydroxyurea in human plasma.

An assay using reversed-phase high-performance liquid chromatography with electrochemical detection was developed to measure hydroxyurea in plasma at concentrations suitable for pharmacokinetic studies. The sample preparation is simple, the analysis rapid and assays can be batched. The between-run precision is excellent (coefficient of variation = 2.8-4.5%) and the limit of detection is 0.02 mmol/l. Preliminary studies have shown that the method is suitable for pharmacokinetic studies.     

Determination of the antiallergenic agent, N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H- pyrido [2,1-b]quinazoline-8-carboxamide, in plasma by reversed-phase high-performance liquid chromatographic analysis using fluorometric detection

A rapid, sensitive and selective high-performance liquid chromatographic (HPLC) assay was developed for the determination of the antiallergenic compound N-[4-(1H-imidazol-1-yl)butyl]-2-(1-methylethyl)-11-oxo-11H-pyrido[ 2,1-b] quinazoline-8-carboxamide (I), and its major metabolite, 2-(1-methylethyl)-11-oxo-11H-pyrido[2,1-b] quinazoline-8-carboxylic acid (I-A), in plasma. The assay involves precipitation of the plasma proteins with acetonitrile--methanol (9:1), followed by the analysis of an aliquot of the protein-free filtrate by reversed-phase ion-pair HPLC with fluorescence detection for quantitation. The analogous compound, N-[6-(1H-imidazol-1-yl)hexyl]-2-(1-methylethyl)-11-oxo-11H-pyrido [2,1-b]-quinazoline-8-carboxamide (II), is used as the internal standard. The overall recovery of compounds I and I-A from plasma is 107.0 +/- 8.6% and 107.0 +/- 10.0%, respectively. The sensitivity limits of quantitation are 20 ng of I, and 10 ng of I-A per ml of plasma using a 0.5-ml aliquot. The assay was used to monitor the plasma concentrations of I and of I-A in a dog following a 5 mg/kg intravenous infusion of I . 2HCl, a 10 mg/kg oral dose of I . 2HCl and of metabolite I-A.     

Simultaneous quantification of budesonide and its two metabolites, 6beta-hydroxybudesonide and 16alpha-hydroxyprednisolone,in human plasma by liquid chromatography negative electrospray ionization tandem mass spectrometry

A sensitive, rapid and selective liquid chromatography negative electrospray ionization tandem mass spectrometry [LC-(-)ESI-MS-MS] method has been developed and validated for the simultaneous quantification of budesonide (BUD) and its major metabolites, 6beta-hydroxybudesonide (OH-BUD) and 16alpha-hydroxyprednisolone (OH-PRED) in human plasma. The method was validated over a linear range from 0.1 to 10 ng/mL for all three analytes using a solid-phase extraction procedure with 9-fluoro-hydrocortisone as the internal standard. The between-day and within-day coefficients of variation for all compounds were < or =20% at the concentrations of lower limit of quantification and < or =15% at other quality control concentrations. The utility of this assay was demonstrated by monitoring BUD, OH-BUD and OH-PRED plasma concentrations in one healthy subject for 24 h following a 3 mg oral dose of budesonide, administered as a pH modified release capsule (Budenofalk) to healthy volunteers.     

Trace level quantification of deuterated 17beta-estradiol and estrone in ovariectomized mouse plasma and brain using liquid chromatography/tandem mass spectrometry following dansylation reaction

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method coupled with dansylation was developed for the simultaneous quantification of exogenously administered deuterated 17beta-estradiol-d4 (E2-d4) and its metabolite, estrone-d4 (E1-d4), in mouse plasma and brain homogenates. The dansylation reaction was simple, fast, and sensitive, and a lower limit of quantification of 50 pg/mL was achieved by using 50 microL of mouse plasma. Interference from endogenous 17beta-estradiol and estrone in plasma and brain samples was minimized by the use of deuterated-E2 as well by utilizing ovariectomized (OVX) mice. The recovery of dansylated derivative exceeded 83% and the reaction was completed within approximately 3 min. The intra- and inter-day assay precision were better than 12.9% and assay accuracy ranged between 92-104% for E1-d4 and E2-d4 in plasma, respectively. The absorption of E2-d4 at both 1 and 3 mg/kg P.O. was rapid, reaching peak plasma concentrations (Cmax) at 5 min post-dose that was the earliest time point obtained, and were 1.1 and 13.8 ng/mL, respectively; the Cmax values for the estrone metabolite, E1-d4, were 1.1 and 43.2 ng/mL, respectively. The area-under-the-plasma-time curve (AUC(0-2 h)) values were determined to be 0.65 and 2.90 ng. h/mL for E2-d4 and 0.77 and 6.74 ng. h/mL for E1-d4, respectively, at 1 and 3 mg/kg. The mean brain-to-plasma ratio for E1-d4 and E2-d4 after P.O. administration of E2-d4 to the OVX mice at 1 and 3 mg/kg indicated that both E1-d4 and E2-d4 were present in the brain as well as in the circulation.     

Determination of aranidipine and its active metabolite in human plasma by liquid chromatography/negative electrospray ionization tandem mass spectrometry

A simple, sensitive and rapid high-performance liquid chromatography/negative electrospray ionization tandem mass spectrometry method was developed and validated for the assay of aranidipine (AR) and its active metabolite (AR-M) in human plasma. Following a liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reversed-phase column and analyzed by mass spectrometry in the multiple reaction monitoring mode using the respective [M-H]- ions, m/z 387.0 --> 164.0 for AR, m/z 389.1 --> 208.1 for AR-M and m/z 359.0 --> 121.8 for the internal standard. The assay exhibited a linear dynamic range of 0.02-10 ng x mL(-1) for AR and 0.2-100 ng x mL(-1) for AR-M in human plasma. The limits of quantitation were 0.02 ng x mL(-1) for AR and 0.2 ng x mL(-1) for AR-M. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 2.8 min for each sample exhibited its high-throughout analysis ability. The validated method can be applied to analyze human plasma samples for pharmacokinetic studies.     

Stereoselective separation and determination of triadimefon and triadimenol in wheat, straw, and soil by liquid chromatography-tandem mass spectrometry

A sensitive and rapid analytical method was developed for simultaneous determination of triadimefon (TF) and triadimenol (TN) stereoisomers in wheat, straw, and soil by liquid chromatography coupled with triple quadrupole mass spectrometry (LC-MS/MS). The direct enantioseparation of TF and TN was performed on a Lux cellulose-1 column packed with cellulose-tris-(3,5-dimethylphenylcarbamate). The effects of mobile-phase composition on the separation were investigated and stereoisomeric elution orders were confirmed with a polarimeter detector. The pesticides were extracted from samples with acetonitrile and cleaned up by solid-phase extraction or activated carbon. Based on the developed stereoselective LC-MS/MS method, for TF and TN stereoisomers, good linearities were obtained over the concentration range of 0.003-4 mg/L; recoveries were 84.2-102.7% in wheat, 84.0-104.0% in straw, and 85.2-106.8% in soil at spiked concentrations of 0.007-2.0 mg/kg; intra-day and inter-day assay precisions were below 12.2%. Limits of detection (LODs) and limits of quantification (LOQs) in wheat, straw, and soil were 0.001-0.005 mg/kg and 0.007-0.02 mg/kg, respectively. Finally, the method was successfully applied to detect TF and TN stereoisomers in wheat, straw, and soil samples from residual trials in farm.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10 micrometer muBondapak phenyl column with an eluting solvent of water--methanol--1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(D-(-)-alpha-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 +/- 6.3% (S.D.) in the concentration ranges of 0.1-20 microgram per 0.2 ml of plasma with a limit of detection equivalent to 0.5 microgram/ml plasma. The urine assay was validated over a concentration range of 0.025-5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 microgram/ml) using a 0.1-ml urine specimen per assay. The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Application of thermospray liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry for the identification of cynomolgus monkey and human metabolites of SK & F 101468, a dopamine D2 receptor agonist

A combination of thermospray liquid chromatography-mass spectrometry (LC-MS) and LC MS MS has allowed the structural elucidation of a number of metabolites of 4-[2-(dipropylamino)ethyl]-1,3-dihydro-2H-indol-2-one (SK & F 101468) in monkey urine. By using LC-MS-MS with the third quadrupole (Q3) set up in multiple ion detection (MID) mode, a number of metabolites were subsequently detected in the human urine and plasma samples despite very low dosing regimes. This was achieved with minimal sample preparation, e.g. for the urine sample centrifugation was the only preparative step, in order to remove particulate matter, prior to analysis. The good signal-to-noise ratio obtained for the human samples, using LC MS MS with Q3 set up for MID, raised the possibility of a LC-MS-MS quantitative assay. As a result, the detection limit of this method for SK&F 101468 when dissolved in methanol was determined to be in the region of 20 pg on column.     

Determination of human plasma protein binding of baicalin by ultrafiltration and high-performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.     

Pharmacokinetics of Azalomycin F,a Natural Macrolide Produced by Streptomycete Strains,in Rats

As antimicrobial resistance has been increasing, new antimicrobial agents are desperately needed. Azalomycin F, a natural polyhydroxy macrolide, presents remarkable antimicrobial activities. To investigate its pharmacokinetic characteristics in rats, the concentrations of azalomycin F contained in biological samples, in vitro, were determined using a validated high-performance liquid chromatography–ultraviolet (HPLC-UV) method, and, in vivo, samples were assayed by an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method. Based on these methods, the pharmacokinetics of azalomycin F were first investigated. Its plasma concentration-time courses and pharmacokinetic parameters in rats were obtained by a non-compartment model for oral (26.4 mg/kg) and intravenous (2.2 mg/kg) administrations. The results indicate that the oral absolute bioavailability of azalomycin F is very low (2.39 ± 1.28%). From combinational analyses of these pharmacokinetic parameters, and of the results of the in-vitro absorption and metabolism experiments, we conclude that azalomycin F is absorbed relatively slowly and with difficulty by the intestinal tract, and subsequently can be rapidly distributed into the tissues and/or intracellular f of rats. Azalomycin F is stable in plasma, whole blood, and the liver, and presents plasma protein binding ratios of more than 90%. Moreover, one of the major elimination routes of azalomycin F is its excretion through bile and feces. Together, the above indicate that azalomycin F is suitable for administration by intravenous injection when used for systemic diseases, while, by oral administration, it can be used in the treatment of diseases of the gastrointestinal tract.     

Automated-extraction, high-performance liquid chromatographic method and pharmacokinetics in rats of a highly A2-selective adenosine agonist, CGS 21680.

CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine, I) is a highly A2-selective (A2/A1 = 140), high-affinity adenosine agonist. A method has been devised to extract the compound from biological matrices with automated solid-phase extraction using C18 bonded silica columns. This is followed by reversed-phase, paired-ion chromatography on a Supelco LC-18-S column with fluorescence detection. The limit of quantitation is 5 ng/ml, but 1 ng/ml (five times the signal-to-noise ratio) can readily be detected. Tritium-labeled compound was used to study the pharmacokinetics in rats. After an intravenous dose of 0.3 mg/kg, biphasic elimination kinetics were observed for parent I, characterized by half-lives of 1.8 min (distribution) and 15 min (elimination). The volume of distribution in the terminal phase (V beta) was low (0.27 l/kg) and plasma clearance was moderate (0.83 l/kg/h). Although the compound was rapidly absorbed (mean Tmax = 13 min), low concentrations (mean Cmax = 94 ng/ml) were observed after an oral dose of 3.0 mg/kg, and bioavailability was only approximately 1.4%. Radioactivity persisted in plasma longer than parent compound after either dose, but levels were too low for isolation and structure identification of drug-derived compounds.     

Simultaneous determination of lamotrigine, zonisamide, and carbamazepine in human plasma by high-performance liquid chromatography

A high-performance liquid chromatography assay with ultraviolet detection was developed for the simultaneous determination of the anti-epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide and the internal standard chloramphenicol were extracted from serum or plasma using liquid-liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 1-30 microg/mL for lamotrigine, 2-20 microg/mL for carbamazepine, and 1-40 microg/mL for zonisamide. Within- and between-run precision studies demonstrated coefficient of variation <10% at all tested concentrations. Other anti-epileptic medications tested did not interfere with the assay. The method is appropriate for determining lamotrigine, carbamazepine and zonisamide serum or plasma concentrations for therapeutic monitoring.     

Relationship between the clinical effects of berberine on severe congestive heart failure and its concentration in plasma studied by HPLC.

It has been reported that berberine is valuable for long-term treatment of ventricular premature beats (VPBs) and leads to a decrease in mortality for patients with congestive heart failure (CHF). In order to improve its therapeutic value and reduce its side effects, it is necessary to study the relationship between its activity and plasma concentration in patients with CHF. Patients with CHF were treated with conventional therapy for 2 weeks. Immediately after the data from a dynamic electrocardiogram (DCG) and left ventricular ejection fraction (LVEF) were obtained, 1.2 g/day of oral berberine was given. After 2 weeks of berberine therapy, the DCG data and LVEF were reassessed and the plasma berberine concentration was measured by HPLC. Plasma samples were pretreated by extraction with chloroform. Berberine in all samples was determined using a mu Bondapak C(18) column, a mobile phase of acetonitrile:0.02 mol/L phosphoric acid (45:55, v/v), and a UV detector at 346 nm. The mean recovery was 96.5%. The linear range was 40-1600 ng/mL. The detection limit for berberine in plasma was 0. 4 ng. The decrease in frequency and complexity of VPBs and the increase in LVEF in patients with plasma berberine concentrations higher than 0.11 mg/L (n = 31, group B) were more significant than at concentrations lower than 0.11 mg/L (p < 0.01 vs p < 0.05).