An HPLC method with diode array detector for the simultaneous quantification of chloroquine and desethylchloroquine in plasma and whole blood samples from Plasmodium vivax patients in Vietnam,using quinine as an internal standard

A sensitive, simple method for quantification of chloroquine (CQ) and desethylchloroquine (MCQ) in whole blood and plasma from Plasmodium vivax patients has been developed using HPLC with diode array detection (DAD). Solid‐phase extraction on Isolute‐96‐CBA was employed to process 100 μL of plasma/whole blood samples. CQ, MCQ and quinine were separated using a mobile phase of phosphate buffer 25 mm , pH 2.60–acetonitrile (88:12, v/v) with 2 mm sodium perchlorate on a Zorbax SB‐CN 150 × 4.6 mm, 5 μm column at a flow rate of 1.2 mL/min, at ambient temperature in 10 min, with the DAD wavelength of 343 nm. The method was linear over the range of 10–5000 ng/mL for both CQ and MCQ in plasma and whole blood. The limit of detection was 4 ng/mL and limit of quantification was 10 ng/mL in both plasma and blood for CQ and MCQ. The intra‐, inter‐ and total assay precision were <10% for CQ and MCQ in plasma and whole blood. In plasma, the accuracies varied between 101 and 103%, whereas in whole blood, the accuracies ranged from 97.0 to 102% for CQ and MCQ. The method is an ideal technique with simple facilities and instruments, bringing about good separation in comparison with previous methods. © 2016 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd     

Gas chromatographic assay for the new antitumor agent sulfamic acid diester (NSC 329680) and its stability in buffer, blood and plasma

A sensitive gas chromatographic assay with electron-capture detection has been developed for sulfamic acid diester (sulfamic acid 1,7-heptanediyl ester, NSC 329680) based on its conversion to 1,7-diiodoheptane in the presence of excess sodium iodide. The assay is linear up to 1 microgram/ml sulfamic acid diester and has a lower limit of detection of 25 ng/ml from 0.5 ml plasma. The coefficient of variation of the assay is 6.4% at 1 microgram/ml and 8.0% at 100 ng/ml. Sulfamic acid diester is relatively stable in 0.9% sodium chloride and 0.1 M sodium phosphate buffers, pH 7.0-9.0, with half-lives greater than 38 h. The major breakdown product of sulfamic acid diester is sulfamic acid 1,7-heptane-monoyl ester. When added to whole blood sulfamic acid diester shows concentration-dependent breakdown. At 50 and 100 micrograms/ml sulfamic acid diester, the half-time in whole blood is 6.9 h and 65% of the drug is sequestered by the blood cells. At 10 micrograms/ml sulfamic acid diester in blood, there is no detectable breakdown of the drug over 24 h and all of the drug is sequestered by the blood cells. Protein binding of sulfamic acid diester in human plasma is 82% at 10 micrograms/ml and 68% at 100 micrograms/ml.     

Validation of liquid chromatography-electrospray ionization ion trap mass spectrometry method for the determination of mesocarb in human plasma and urine

Mesocarb metabolism in humans is the target of this investigation. A high-performance liquid chromatographic (LC) method with electrospray ionization (ESI)-ion trap mass spectrometric (MS) detection ion trap "SL" for the simultaneous determination of mesocarb and its metabolites in plasma and urine is developed and validated. Ten metabolites and the parent drug are detected in human urine, and only four in human plasma, after the administration of a single oral dose of 10 mg of mesocarb (Sydnocarb, two 5-mg tablets). Seven of this metabolites have been found for the first time. The confirmation of the results and identification of all the metabolites except amphetamine is performed by LC-MS, LC-MS-MS, and LC-MS3. In the case of doping analysis, the reliable detection time for mesocarb (long-life dihydroxymesocarb metabolites of mesocarb) is approximately 10-11 days after the administration of the drug, which is a significant increase over the existing data. The detection of amphetamine in plasma and urine is made using simple flow-injection analysis without a chromatographic separation. The addition-calibration method is used with plasma and urine. The mean recoveries from plasma are 49.2% and 57.4% for mesocarb concentrations of 33.0 and 66.0 ng/mL, respectively, whereas the recoveries from human urine are 76.9% and 81.4% for concentrations of 1 and 2 ng/mL, respectively. Calibration curves (using an internal standard method) are linear (r2>0.9969) for concentrations 0.6 to 67 ng/mL and from 0.05 to 5 ng/mL in plasma and urine, respectively. Both intra- and interassay precision of plasma control samples at 3, 40, and 55 ng/mL are lower than 6.2%, and the concentrations do not deviate for more than -3.4% to 7.3% from their nominal values. In urine, intra- and interassay precision of control samples at 0.08, 1.5, and 3.0 ng/mL is lower than 14.1%, with concentrations not deviating for more than -11.3% to 13.7% from their nominal values. The plasma disappearance curve of the parent drug is obtained. The major pharmacokinetic parameters are calculated.     

Determination of N-methyl-4-isoleucine-cyclosporin (NIM811) in human whole blood by high performance liquid chromatography-tandem mass spectrometry

A liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of N-methyl-4-isoleucine-cyclosporin (NIM811) was developed and validated over the concentration range 1-2500 ng/mL in human whole blood using a 0.05 mL sample volume. NIM811 and the internal standard, d(12)-cyclosporin A (d(12)-CsA), were extracted from blood using MTBE via liquid-liquid extraction. After evaporation of the organic solvent and reconstitution, a 10 microL aliquot of the resulting extract was injected onto the LC-MS/MS system. Chromatographic separation of NIM811 and internal standard was performed using a Waters Symmetry RP-8 (50 x 4.6 mm, 3 microm particle size) column. The mobile phase consists of 10 mm ammonium acetate in water (A) and acetonitrile (B), with 45% B from 0 to 0.2 min, 45 to 85% B from 0.2 to 0.8 min and 85% B from 0.8 to 2.2 min. The total run time was 3.5 min with a flow rate of 0.8 mL/min. The method was validated for sensitivity, linearity, reproducibility, stability, dilution integrity and recovery. The precision and accuracy of quality control samples at low (2.00 ng/mL), medium (20.0 and 400 ng/mL) and high (2000 ng/mL) concentrations were in the range 1.1-4.3% relative standard deviation (RSD) and -2.5-10.0% (bias), respectively, from three validation runs. The method has been used to measure the exposure of NIM811 in human subjects.     

A sensitive high performance liquid chromatography microassay for bemoradan, a novel cardiotonic agent, in plasma/serum using a semi-automatic liquid/solid extraction sample preparation system--AASP

A high performance liquid chromatographic method for the measurement of bemoradan levels in plasma/serum is described. This method uses Varian's AASP (Varian Associates, Sunnyvale, CA, USA), a semi-automatic liquid/solid extraction sample preparation system. It requires only small volumes of plasma/serum samples (0.2-1 mL) and needs no organic solvent for sample preparation. The mean recovery of bemoradan at plasma or serum concentrations of 0.5-100 ng/mL is 82%. The assay has a detection limit of 0.5 ng/mL (when 1 mL of plasma/serum is used) and is linear in the concentration range 0.5-500 ng/mL.     

Washing-free chemiluminescence immunoassay for rapid detection of cardiac troponin Ⅰ in whole blood samples

Chemiluminescence immunoassay(CLⅠA) has always been a great challenge in detecting cardiac troponin Ⅰ(c Tn Ⅰ) in whole blood samples without centrifugation because of the interference of red blood cells and low sensitivity. Ⅰn this study, the antigens and erythrocytes in the blood were captured by the antibodies immobilized on the magnetic particles, recognized by another biotinconjugated c Tn Ⅰ antibody and detected by streptavidin/acridine aster-conjugated polychloromethylstyrene microspheres(...     

Liquid chromatographic method for the determination of sirolimus in blood using electrochemical detection

Therapeutic drug monitoring of sirolimus (rapamycin) is important for immunosuppressive therapy in solid organ transplantation. We have developed a simple and reliable method for determining blood concentrations of sirolimus using reversed-phase HPLC with electrochemical detection (ECD). The E(2) potential was set at +900 mV. The potential of guard cell was set at +950 mV and that of the E(1) cell at +400 mV. The method was linear for a concentration range of 1-50 ng/mL when 0.5 mL blood was used. The correlation coefficients of all standard curves were greater than or equal to 0.999. The limit of detection was 0.5 ng/mL. The inter-assay precision ranged from 3.22 to 7.48%, and the coefficient of variation (CV) for a quality control sample at 10 ng/mL was 7.48% with a bias of 8.4% from the target value. The intra-assay precision ranged from 0.72 to 3.71%, and the CV for a quality control sample at 10 ng/mL was 0.72% with a bias of 6.8% from the target value. In a solid organ transplant recipient, trough concentrations of sirolimus were well within the analytic range of the HPLC/ECD procedure. The method described here is suitable for management of sirolimus therapy in solid organ transplantation.     

A simple and sensitive HPLC-fluorescence method for quantification of MDMA and MDA in blood with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label

A sensitive high-performance liquid chromatographic method with fluorescence detection to determine 3,4-methylenedioxymethamphethamine (MDMA) and 3,4-methylenedioxyamphethamine (MDA) in human and rat whole blood or plasma samples was developed by using 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl) as a label. MDMA and MDA in a small amount of blood sample (ca 100 microL) were extracted by liquid-liquid extraction with ethyl acetate, and were derivatized with DIB-Cl under mild conditions (10 min at room temperature). A good separation of DIB-derivatives could be achieved within 45 min using a commercially available ODS column with an isocratic eluent of 10 mM citric acid-20 mM Na(2)HPO(4) aqueous buffer (pH 4.0)-CH(3)CN-CH(3)OH (50:45:5, v/v/v %). The calibration curves prepared with 1-methyl-3-phenylpropylamine (MPPA) as an internal standard showed good linearity (r = 0.999) with 0.36-0.83 ng/mL detection limit at a signal-to-noise ratio of 3. MDMA and MDA in rat whole blood could be monitored for 6 h after a single administration of MDMA (2.2 mg/kg, i.p.). The pharmacokinetic parameters for MDMA and MDA obtained by triplicate measurements were 426 +/- 23 and 39 +/- 6 ng/mL (C(max)), 20 +/- 5 and 100 +/- 10 min (T(max)), respectively.     

Liquid chromatography/tandem mass spectrometry determination of (4S,2RS)-2,5,5-trimethylthiazolidine-4-carboxylic acid, a stable adduct formed between D-(-)-penicillamine and acetaldehyde (main biological metabolite of ethanol), in plasma, liver and brain rat tissues

Acetaldehyde, the main biological metabolite of ethanol, is nowadays considered to mediate some ethanol-induced effects. Previous studies on alcohol effect attenuation have shown that D-(-)-penicillamine (3-mercapto-D-valine), a thiol amino acid, acts as an effective agent for the inactivation of acetaldehyde. In the study reported here, laboratory rats were treated with ethanol and D-(-)-penicillamine at different doses looking for the interaction (in vivo) of D-(-)-penicillamine with metabolically formed acetaldehyde following a condensation reaction to form the stable adduct (4S,2RS)-2,5,5-trimethylthiazolidine-4-carboxylic acid (TMTCA). A novel and rapid procedure based on liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) was developed for quantification and reliable identification of TMTCA in different rat tissues, including plasma, liver and brain. Firstly, plasma was obtained from whole blood. Then, proteins were precipitated from plasma, brain and liver extracts with acetonitrile and the clarified extracts diluted 10-fold. A 20 microL aliquot of the final extracts was then analyzed using an Atlantis C18 5 microm, 100x2 mm column which was connected to the electrospray source of a LC/triple quadrupole mass spectrometer. The analyte was detected in positive ion mode acquiring four MS/MS transitions in selected reaction monitoring (SRM) mode.The method has been validated and it has proved to be fast, reliable and sensitive. The accuracy and precision were evaluated by means of recovery experiments from plasma, liver and brain samples fortified at two concentration levels obtaining satisfactory recoveries in all cases: 95 and 105% in plasma (at 10 and 100 ng/mL, respectively), 79 and 89% in brain (100 and 1000 ng/g), 85 and 99% in liver (100 and 1000 ng/g). Precision, expressed as repeatability, was in all tissues analyzed lower than 17% at the two concentrations tested. The estimated detection limits were 1 ng/mL in plasma, 4 ng/g in brain and 5 ng/g in liver. The limit of quantitation objective (the lowest concentration that was validated with acceptable results) was set up at 10 ng/mL for plasma and 100 ng/g for brain and liver tissue.The reliable identification of the analyte was ensured by the acquisition of four transitions and by their ion abundance ratio measurement. Due to its excellent selectivity and sensitivity, the method developed in this work provides an excellent tool for the specific determination of this cyclic amino acid in biological samples.     

A simple and sensitive HPLC method for quantitation of low phenoprolamine hydrochloride concentrations in human plasma and its pharmacokinetic application

Phenoprolamine hydrochloride is a novel compound that works against a variety of types of hypertension. The purpose of this study was to develop a simple and sensitive high-performance liquid chromatographic method for quantitation of low phenoprolamine hydrochloride concentrations in human plasma and to apply it to pharmacokinetic study. The procedure involved extraction of the drug and clonidine (internal standard) from the plasma using diethyl ether. Chromatographic separations were carried out on a 4.6 x 200 mm Hypersil silica column with UV detection at 230 nm. The isocratic mobile phase, 1% ammonium acetate (pH 5.4) and methanol (0.3:99.7, v/v), was run at 1 mL/min. Extraction recovery was 84% for phenoprolamine hydrochloride at a concentration level of 200 ng/mL, and 76% for clonidine at 200 ng/mL. The method was linear in the concentration range 5-4000 ng/mL with a lower limit of quantitation of 5 ng/mL for phenoprolamine hydrochloride. Inter- and intra-day coefficients of variation were less than 10%. The validated method was successfully applied to a pharmacokinetic study in human after an oral administration of the drug, and the pharmacokinetic parameters are presented.     

Head-column field-amplified sample stacking in capillary electrophoresis for the determination of cimetidine, famotidine, nizatidine, and ranitidine-HCl in plasma.

In this study, low concentrations of histamine2-receptor (H2-)antagonists were effected across a water plug, with separation taking place in a binary buffer comprising ethylene glycol and NaH2PO4 (pH 5.0), and detection at 214 nm. Liquid-liquid extraction with ethyl acetate- isopropanol is shown to provide extracts that are sufficiently clean. The calibration curves were linear over a concentration range of 0.1-2.00 microg/mL cimetidine, 0.2-5.0 microg/mL ranitidine-HCl, 0.3-5.0 microg/mL nizatidine, and 0.1-3.0 microg/mL famotidine. Mean recoveries were > 82%, while the intra- and interday relative standard deviations (RSDs) and relative errors (REs) were all < 13%. The method is sensitive with a detection limit of 3 ng/mL cimetidine, 30 ng/mL ranitidine HCl, 50 ng/mL nizatidine and 10 ng/mL famotidine (S/N = 3, electric-driven injection 90 s). This newly developed capillary electrophoresis (CE) method was applied for the determination of analytes extracted from plasma taken from a volunteer dosing a cimetidine, ranitidine, and nizatidine tablet simultaneously. These three H2-antagonists can be detected in real samples by this method, excluding the low dosing of famotidine tablet.     

Quantification of clenbuterol in equine plasma,urine and tissue by liquid chromatography coupled on-line with quadrupole time-of-flight mass spectrometry

Clenbuterol (CBL) is a potent beta(2)-adrenoceptor agonist used for the management of respiratory disorders in the horse. The detection and quantification of CBL can pose a problem due to its potency, the relatively low dose administered to the horse, its slow clearance and low plasma concentrations. Thus, a sensitive method for the quantification and confirmation of CBL in racehorses is required to study its distribution and elimination. A sensitive and fast method was developed for quantification and confirmation of the presence of CBL in equine plasma, urine and tissue samples. The method involved liquid-liquid extraction (LLE), separation by liquid chromatography (LC) on a short cyano column, and pseudo multiple reaction monitoring (pseudo-MRM) by electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI-QTOF-MS/MS). At very low concentrations (picograms of CBL/mL), LLE produced better extraction efficiency and calibration curves than solid-phase extraction (SPE). The operating parameters for electrospray QTOF and yield of the product ion in MRM were optimized to enhance sensitivity for the detection and quantification of CBL. The quantification range of the method was 0.013-10 ng of CBL/mL plasma, 0.05-20 ng/0.1 mL of urine, and 0.025-10 ng/g tissue. The detection limit of the method was 13 pg/mL of plasma, 50 pg/0.1 mL of urine, and 25 pg/g of tissue. The method was successfully applied to the analysis of CBL in plasma, urine and various tissue samples, and in pharmacokinetic (PK) studies of CBL in the horse. CBL was quantified for 96 h in plasma and 288 h in urine post-administration of CLB (1.6 micro g/kg, 2 x daily x 7 days). This method is useful for the detection and quantification of very low concentrations of CBL in urine, plasma and tissue samples.     

A high-performance liquid chromatography/tandem mass spectrometry method for the determination of artemisinin in rat plasma

Artemisinin is a widely used antimalarial drug. To evaluate the pharmacokinetics of artemisinin in rats, a sensitive and specific liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed and validated for the determination of artemisinin in rat plasma. For detection, a Sciex API 4000 LC/MS/MS instrument with an electrospray ionization (ESI) TurboIonSpray inlet in the positive ion multiple reaction monitoring (MRM) mode was used to monitor precursor ([M+NH4]+) --> product ions of m/z 300.4 --> 209.4 for artemisinin and m/z 316.4 --> 163.4 for artemether, the internal standard (IS). The plasma samples were pretreated by a simple liquid-liquid extraction with ether. The standard curve was linear (r > 0.99) over the artemisinin concentration range of 1.0-200.0 ng/mL in plasma. The method had a lower limit of quantification of 1.0 ng/mL for artemisinin in 100 microL of plasma, which offered a satisfactory sensitivity for the determination of artemisinin. The intra- and inter-day precisions were measured to be within +/-5.3% and accuracy between -2.6% and 1.2% for all quality control samples, lower limit of quantification and upper limit of quantification samples. The extraction recoveries of artemisinin and the IS were 95.4 +/- 4.5% and 92.8 +/- 3.9%, respectively. This present method was successfully applied to the characterization of the pharmacokinetic profile of artemisinin in rats after oral administration.     

Determination of ecgonine and seven other cocaine metabolites in human urine and whole blood by ultra-high-pressure liquid chromatography–quadrupole time-of-flight mass spectrometry

Ecgonine is suggested to be a promising marker of cocaine (COC) ingestion. A combined mass spectrometry (MS) and tandem MS (MS/MS) method was developed to simultaneously determine ecgonine and seven other metabolites of cocaine in human urine and whole blood with ultra-high-pressure liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. The compounds were extracted from as little as 100 μL of sample by solid-phase extraction with a 96-well μElution solid-phase extraction plate. The protonated molecules or fragment ions at accurate mass acquired in MS mode were used to quantify specific analytes, following by dedicated MS/MS identification. The assay was linear in the range from 5 to 50-100 ng/mL for urine samples, except for ecgonine methyl ester (10-200 ng/mL) and ecgonine (40-400 ng/mL), and was linear from 1-2 to 50 ng/mL for whole blood samples, except for ecgonine methyl ester (20-1,000 ng/mL) and ecgonine (40-2,000 ng/mL). The correlation coefficients were all greater than 0.99. The limits of detection ranged from 0.2 to 16 ng/mL, and the lower limits of quantification ranged from 1 to 40 ng/mL. The repeatability and intermediate precision were 18.1 % or less. The accuracy was in the range from 80.0 to 122.9 %, process efficiencies were in the range from 8.6 to 177.4 %, matrix effects were in the range from 28.7 to 171.0 %, and extraction recoveries were in the range from 41.0 to 114.3 %, except for ecgonine (12.8 % and 9.3 % at low and high concentrations, respectively). This method was highly sensitive in comparison with previously published methods. The validated method was successfully applied to the analysis of real samples derived from forensic cases, and the results verified that, on the basis of data from four positive samples, ecgonine is a promising marker of cocaine ingestion.

Development of a rapid and specific assay for detection of busulfan in human plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A sensitive and specific assay for detection of busulfan in human plasma was developed. The assay is based on rapid isolation of busulfan by liquid-liquid extraction with ethyl acetate, and detection by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry. 1,6-Bis(methanesulfonyloxy)hexane, a synthesized analogue of busulfan, was used as the internal standard (IS). The acquisition was performed in the multiple reaction monitoring mode; busulfan and the IS were detected with no interferences from plasma matrix. The method was linear over the range 5-2500 ng mL(-1), with r2 > 0.99 and a run time of only 3.5 min. The intra- and inter-assay precisions were in the ranges 2.1-11.9% and 3.2-10.1%, respectively, and the intra- and inter-assay accuracies were 92.2-107.6% and 94.7-104.1%, respectively. The absolute recoveries were 82.0% (20 ng mL(-1)), 90.6% (1000 ng mL(-1)) and 80.0% (2000 ng mL(-1)) for busulfan, and 89.1% for the IS (1000 ng mL(-1)). The limits of detection and quantification were 2 and 5 ng mL(-1), respectively. The validated method was successfully applied to analyze plasma samples obtained from six adults receiving doses of 1 mg kg(-1) in a conditioning regimen prior to bone marrow transplantation. A marked intra-patient variation in busulfan concentrations during the steady state was observed, which limits the application of pharmacokinetic modeling and suggests that continuous therapeutic monitoring is necessary for adequate individualized dosing. In this regard, the present assay brings important advantages relative to other methods described in the literature, i.e., it is highly specific and simple to perform, with a rapid chromatographic run time (3.5 min), and the whole procedure can be completed in 4-5 h, which would permit dose corrections after the third dose allowing earlier and better dosing adjustments towards the target level of busulfan.     

High performance liquid chromatography/electrospray tandem mass spectrometry for phenothiazines with heavy side chains in whole blood

Eleven phenothiazine derivatives with heavy side chains contained in human whole blood have been analyzed by high-performance liquid chromatography (HPLC)/electrospray (ES) tandem mass spectrometry (MS). All compounds gave the base peaks due to [M + 1](+) by HPLC/ES single MS. The product ions formed from each quasi-molecular ion by HPLC/ES tandem MS showed the base peaks due to side chains liberated. The mass chromatography of HPLC/ES tandem MS showed much higher sensitivity than that of HPLC/ES single MS for phenothiazines spiked to whole blood. Therefore, regression equations, detection limits, recovery rates and reproducibility were studied for thiethylperazine, clospirazine and flupentixol spiked to human whole blood by means of mass chromatography of HPLC/ES tandem MS. The three compounds showed good linearity in the range of 2-40 ng/mL with a detection limit of about 0.5 ng/mL. Recoveries of the three compounds spiked to whole blood (2 and 8 ng added to 1 mL whole blood) were 43.4-72.5 %; the coefficients of intraday and interday variations were 3.7-9.3 and 12.6-17.9 %, respectively. Thiethylperazine, clospirazine and flupentixol in whole blood could actually be determined with sufficient sensitivity 3 and 6 h after oral administration of 5-10 mg of each compound in a volunteer.     

Development and validation of a liquid chromatography and ion spray tandem mass spectrometry method for the quantification of artesunate, artemether and their major metabolites dihydroartemisinin and dihydroartemisinin-glucuronide in sheep plasma

Recently, promising fasciocidal activities of artesunate and artemether were described in rats and sheep. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify artesunate, artemether and their metabolites dihydroartemisinin and dihydroartemisinin-glucuronide in sheep plasma. Protein precipitation with methanol was used for sample workup. Reversed-phase high-performance liquid chromatography (HPLC) was performed using an Atlantis C18 analytical column with a mobile phase gradient system of ammonium formate and acetonitrile. The analytes were detected by MS/MS using selected reaction monitoring (SRM) with electrospray ionisation in the positive mode (transition m/z 267.4 → 163.0). The analytical range for dihydroartemisinin, dihydroartemisinin-glucuronide and artesunate was 10-1000 ng/ml and for artemether 90-3000 ng/ml with a lower limit of quantification of 10 and 90 ng/ml, respectively. Inter- and intra-day accuracy and precision deviations were < 10%. Consistent relative recoveries (60-80%) were observed over the investigated calibration range for all analytes. All analytes were stable in the autosampler for at least 30 h (6 °C) and after three freeze and thaw cycles. The validation results demonstrated that the LC-MS/MS method is precise, accurate and selective and can be used for the determination of the artemisinins in sheep plasma. The method was applied successfully to determine the pharmacokinetic parameters of artesunate and its metabolites in plasma of intramuscularly treated sheep.     

Concentration profiles of cocaine, pyrolytic methyl ecgonidine and thirteen metabolites in human blood and urine: determination by gas chromatography-mass spectrometry

When cocaine is smoked, a pyrolytic product, methyl ecgonidine (anhydroecgonine methyl ester), is also consumed with the cocaine. The amount of methyl ecgonidine formed depends on the pyrolytic conditions and composition of the illicit cocaine. This procedure describes detection of cocaine and 10 metabolites--cocaethylene, nor-cocaine, nor-cocaethylene, methyl ecgonine, ethyl ecgonine, benzoylecgonine, nor-benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine and ecgonine--in blood and urine. In addition, the detection of pyrolytic methyl ecgonidine and three metabolites--ecgonidine (anhydroecgonine), ethyl ecgonidine (anhydroecgonine ethyl ester) and nor-ecgonidine (nor-anhydroecgonine)--are included. The newly described metabolites, ethyl ecgonidine and nor-ecgonidine, were synthesized and characterized by gas chromatography-mass spectrometry (GC-MS). All 15 compounds were extracted from 3 mL of blood or urine by solid-phase extraction and identified by a GC-MS method. The overall recoveries were 49% for methyl ecgonine, 35% for ethyl ecgonine, 29% for ecgonine and more than 83% for all other drugs. The limits of detection were between 0.5 and 4.0 ng/mL except for ecgonine, which was 16 ng/mL. Linearity for each analyte was established and in all cases correlation coefficients were 0.9985-1.0000. The procedure was applied to examine the concentration profiles of analytes of interest in post-mortem (PM) blood and urine, and in urine collected from living individuals (LV). These specimens previously were shown to be positive for the cocaine metabolite, benzoylecgonine. Ecgonidine, the major metabolite of methyl ecgonidine, was present in 77% of PM and 88% of the LV specimens, indicating smoking as the major route of cocaine administration. The new pyrolytic metabolites, ethyl ecgonidine and nor-ecgonidine, were present in smaller amounts. The urine concentrations of nor-ecgonidine were 0-163 ng/mL in LV and 0-75 ng/mL in PM specimens. Ethyl ecgonidine was found only in PM urine at concentrations 0-39 ng/mL. Ethanol-related cocaine metabolites, ethyl ecgonine or cocaethylene, were present in 69% of PM and 53% of cocaine-positive LV specimens, implying alcohol consumption with cocaine use. The four major metabolites of cocaine--benzoylecgonine, ecgonine, nor-benzoylecgonine and methyl ecgonine--constituted approximately 88 and 97% of all metabolites in PM and LV specimens, respectively. The concentrations of nor-cocaine and nor-cocaethylene were consistently the lowest of all cocaine metabolites. At benzoylecgonine concentrations below 100 ng/mL, ecgonine was present at the highest concentrations. In 20 urine specimens, benzoylecgonine and ecgonine median concentrations (range) were 54 (0-47) and 418 ng/mL (95-684), respectively. Therefore, detection of ecgonine is advantageous when benzoylecgonine concentrations are below 100 ng/mL.     

Preparation of a Monoclonal Antibody against a Kallikrein-Like Enzyme from Agkistrodon halys pallas Venom and Its Application in a Pharmacokinetic Study

Snake venom contains bioactive materials for drug development, diagnosis, and treatment. After separating and purifying the kallikrein-like enzyme (AHP-Ka) from Agkistrodon halys pallas venom for the first time, a monoclonal antibody against AHP-Ka was prepared and characterized. An indirect sandwich enzyme-linked immunosorbent assay (ELISA) based on the monoclonal antibody was developed and validated for the pharmacokinetic analysis of AHP-Ka in rat plasma. The method was calibrated using rat plasma and 1:100 dilution of plasma was selected to prepare a calibration curve to validate the precision, accuracy, and stability of the ELISA method. A good linear relationship was obtained in a working range from 3.9 ng/mL to 62.5 ng/mL with a limit of detection of 2.94 ng/mL. Intra- and inter-batch precision were less than 10%. The average recovery ranged from 94.6% to 104.4% in rat plasma at the concentrations of 5 ng/mL, 15 ng/mL, and 45 ng/mL, respectively. The ELISA method was successfully used for the pharmacokinetic study of AHP-Ka in Sprague-Dawley rat plasma after intravenous administration. The work is expected to contribute to future preclinical development of AHP-Ka.     

HPLC quantification of metoprolol with solid-phase extraction for the drug monitoring of pediatric patients

Although the analytical literature seems abundant for the determination of metoprolol in human plasma, a method using standard equipment providing a sensitive and simple high-performance liquid chromatographic (HPLC) method for limited blood volume, e.g. where 1 mL of blood in a 1 kg infant equals 70 mL of adult blood volume, has rarely been addressed. Therefore, in 500 microL of plasma, metoprolol was extracted using an internal standard and solid-phase extraction columns. Chromatographic analysis was performed on a Spherisorb C(6) column (5 microm particle size) at ambient temperature and fluorimetric detection with an excitation wavelength of 225 nm, and emission wavelength of 310 nm. The mobile phase [30% acetonitrile and 70% 0.25 m potassium acetate buffer (pH 4)] was pumped with 1 mL/min. Metoprolol recovery was determined at 73.0 +/- 20.5%, and the limit of quantitation was 2.4 ng/mL. Precision values of intra- and inter-assay were below 15.5% and those for accuracy were between 90 and 110%. This method was developed for monitoring and determination of pharmacokinetic parameters of metoprolol in pediatric patients and therefore metoprolol plasma concentrations in a 2-year-old child with ventricular tachycardia are reported. .