New simple liquid chromatographic method for the determination of trimethoprim, sulfadiazine and N4-acetylsulfadiazine in plasma of broilers

A new method for simultaneous quantification of trimethoprim, sulfadiazine and N4-acetylsulfadiazine in plasma of broilers at levels down to 13-16 ng/ml has been developed. Samples were deproteinized with acetonitrile, defatted with hexane, and extracted with dichloromethane. Chromatographic analysis was carried out on a C18 column in the presence of tetrabutylammonium hydrogen sulfate, a competing base, while detection was performed at 240 nm for trimethoprim, and 270 nm for both sulfadiazine and N4-acetylsulfadiazine. Accuracy and precision data showed recoveries and relative standard deviation values better than 87.3% and 3.1%, respectively, for all three analytes. The good analytical characteristics of the method could allow limits of detection in the low ng/ml range to be realised. The method was successfully applied to determine drug concentrations in plasma samples from broilers administered a combination of sulfadiazine and trimethoprim.     

Liquid chromatographic method for the simultaneous determination of cefalexin and trimethoprim in dog plasma and application to the pharmacokinetic studies of a coformulated preparation

A liquid chromatographic method is described for the simultaneous determination of cefalexin and trimethoprim in dog plasma. A simple protein precipitation procedure was adopted for the sample preparation with satisfactory extraction recoveries for both analytes. Chromatographic separation of the analytes was achieved on a C(18) column using a mixture of 2 mol/l formate buffer (pH 3.5), methanol and acetonitrile (22:7:7, v/v/v) containing a 0.002 mol/l sodium dodecyl sulfate as mobile phase and detection was performed at 240 nm. The linearity was obtained over the concentration ranges of 1.0-100.0 microg/ml for cefalexin and 0.5-50.0 microg/ml for trimethoprim. For each level of QC samples including the lower limit of quantification, both inter- and intra-day precisions (R.S.D.) were < or =14.0% for cefalexin and < or =11.4% for trimethoprim, and accuracy (RE) was -1.4% for cefalexin and -3.0% for trimethoprim. The present LC method was successfully applied to the pharmacokinetic studies of coformulated cefalexin dispersible tablets after oral administration to beagle dogs.     

Ion-pair reversed-phase HPLC: assay validation of sodium tanshinone IIA sulfonate in mouse plasma

Sodium tanshinone IIA sulfonate (STS), a hydrophilic ionic substance, is used as a cardiovascular drug. An ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) method for the determination of STS in mouse plasma was initially developed. The assay involved a rapid and simple extraction process and subsequent detection at 271 nm. The retention time for STS was 7.5 min. Based on extracted STS standard mouse plasma at 1.5,10 and 50 microg/ml, the assay precision were 2.7, 2.1 and 1.7% with a mean accuracy of 96.7, 98.5 and 99.4%, respectively. At plasma concentration of 1.5, 50 and 75 microg/ml, the mean recovery of STS were 93.1, 96.3 and 97.5%. The limit of detection (LOD) and limit of quantification (LOQ) for STS was 0.1 microg/ml and 0.5 microg/ml, respectively. Linear responses were observed over a wide concentration range (0.5-100 microg/ml) for STS in mouse plasma. STS can be detected after intravenous administration. This method was performed for the first time in pharmacokinetic studies of STS in the mouse.     

Enantioselective high-performance liquid chromatographic assay for determination of the enantiomers of a new anti-ulcer agent,E3810, in Beagle dog plasma and rat plasma

An enantioselective high-performance liquid chromatographic method for the determination of E3810, a new anti-ulcer agent, in Beagle dog plasma and rat plasma has been developed. After extraction from plasma with ethyl acetate. E3810 enantiomer were measured by reversed-phase high-performance liquid chromatography on a Chiralcel OD-R column. The enantiomers were detected by ultraviolet absorbance detection at 290 nm. The recoveries of E3810 enantiomers and internal standard were greater than 91%. The calibration curves were linear from 0.03 to 20 μg/ml for Beagle dog plasma and from 0.1 to 100 μg/ml for rat plasma. The limits of quantification of both enantiomers were 0.03 μg/ml for Beagle dog plasma and 0.1 μg/ml for rat plasma. The intra- and inter-day accuracy and precision data showed good reproducibility of the method. The assay was applied for the analysis of E3810 enantiomers in plasma after intravenous administration of racemic E3810 to Beagle dogs and rats. This method should be very useful for enantioselective pharmacokinetic studies of E3810.     

Development and validation of an HPLC-UV method for iodixanol quantification in human plasma

Iodixanol is a widely used iso-osmolar contrast medium agent. Similar to iohexol, it can also be a good exogenous marker for the measurement of glomerular filtration rate (GFR). This article describes the development and validation of an HPLC-UV method for quantification of iodixanol in human plasma. Internal standard, iohexol (20 microl, 1 mg/ml), and perchloric acid (30 microl, 20%, v/v) were added to plasma samples (300 microl), followed by neutralization with 10 microl potassium carbonate (5M). Samples were centrifuged and 10 microl of the supernatant was injected onto a C(18) EPS analytical column (3 microm particle size, 150 mm x 4.6 mm). The extraction method yielded >95% recovery for both iodixanol and iohexol. The mobile phase consisted of 0.1% (w/v) sodium formate buffer and acetonitrile. Iohexol and iodixanol peaks were eluted at approximately 5 and 9 min, respectively using a fast gradient method. The assay lower limit of detection was 2.0 microg/ml and lower limit of quantification was 10 microg/ml. The calibration curves, assessed in six replicates, were linear over an iodixanol concentration range of 10-750 microg/ml. Intra- and inter-day accuracy was >95% and precision expressed as % coefficient of variation was <10%. This method is simple, accurate, precise and robust and can potentially be used for iodixanol quantification in large-scale clinical studies.     

Comparison of folate quantification in foods by high-performance liquid chromatography-fluorescence detection to that by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry

A comparison study on folate quantitation was carried out between the recently developed stable isotope dilution assay using liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the frequently used HPLC with fluorimetric detection (LC-FD). By applying LC-MS-MS, spinach, wheat bread, beef, and blood plasma were found to contain 159.2, 19.8, 1.2, and 5.6 microg/100 g total folates, respectively, whereas the respective quantitative data obtained by LC-FD were 95.5, 16.2, 0.7, and 6.8 microg/100 g. In all samples, LC-MS-MS revealed superior selectivity and precision and circumvented the shortcomings of conventional LC techniques, i.e., ambiguous peak assignment as well as high detection limits for 5-formyltetrahydrofolate, 10-formylfolic acid, and folic acid. The affinity chromatography columns used in this study showed excellent cleanup performance and permitted detection limits as low as 0.1, 0.5, 0.1, 0.08, and 0.1 microg/100 g for tetrahydrofolate (H(4)folate), 5-methyl-H(4)folate, 5-formyl-H(4)folate, 10-formylfolate, and pteroylglutamic acid, respectively. Thus, a 10-fold higher sensitivity compared to solid-phase anion-exchange cartridges was achieved. However, affinity chromatography columns revealed a significantly higher affinity toward the natural vitamers than to the racemic isotopomeric standards, which has to be considered when applying the latter in stable isotope dilution assays.     

Liquid chromatographic-tandem mass spectrometric method for the quantitation of huperzine A in dog plasma

A rapid and sensitive LC-MS-MS method for the determination of huperzine A in dog plasma using huperzine B as internal standard has been developed and validated. The analyte and internal standard were extracted from plasma using n-hexane-dichloromethane-2-propanol (300:150:15, v/v/v), chromatographed on a C(18) column (5 microm, 50 mm x 4.6 mm i.d.) with a mobile phase consisting of acetonitrile-methanol-10mM ammonium acetate (35:40:25, v/v/v), and detected using a tandem mass spectrometer with a TurboIonSpray ionization interface. The run time was only 2 min. The assay was linear over the concentration range 0.05-20 ng/ml and intra- and inter-day precision over this range were <5.3% with good accuracy. The limit of detection in plasma was 0.01 ng/ml. The method was successfully applied to define plasma concentration-time curves of huperzine A in dogs after the last dose of an intramuscular injection (10 microg/kg per day for 15 days) of a sustained-release formulation of huperzine A.     

High-performance liquid chromatographic assay validation of Manumycin A in mouse plasma

Manumycin A is a natural antibiotic produced by Streptomyces parvulus that has antineoplastic activity against a variety of human cancers in nude mouse models. We have developed a highly sensitive reverse phase high-performance liquid chromatography (HPLC) method based on ultraviolet (UV) detection for the determination of manumycin A in mouse plasma. Manumycin A was isolated from mouse plasma by solid-phase extraction. A gradient elution of methanol and 0.05 M H(3)PO(4) with 0.2% triethylamine mobile phase was employed and separation was achieved with a C(18) analytical column. Manumycin A was detected by UV absorption at 345 nm. Retention time for manumycin A was 8.9+/-0.2 min. The manumycin A peak was baseline resolved, with the nearest peak at 1.5 min distance and no interfering peaks detected. Inter- and intra-day coefficients of variance were less than 6.1 and 5.1%, respectively. Based on an extracted manumycin A standard plasma sample of 0.25 microg/ml, the assay precision was 99.8% with a mean accuracy of 95.1%. At plasma concentrations of 0.5 and 5 microg/ml, the mean recovery rates of manumycin A were 59.64 and 60.28%, respectively. The lower limit of detection (LLD) for manumycin A was 0.1 microg/ml in mouse plasma. The lower limit of quantification (LLQ) for manumycin A was 0.125 microg/ml. Results of the stability study indicated that when frozen at -80 degrees C, manumycin A was stable in mouse plasma for up to 2 weeks. This method is useful in quantification of manumycin A in mouse plasma for clinical pharmacology studies in mice.     

Validated HPLC method for the determination of gabapentin in human plasma using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene and its application to a pharmacokinetic study

A rapid, sensitive and accurate high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of gabapentin in human plasma. Gabapentin was quantified using pre-column derivatization with 1-fluoro-2,4-dinitrobenzene following protein precipitation of plasma with acetonitrile. Amlodipine was used as internal standard. The chromatographic separation was carried out on a Nova-Pak C(18) column using a mixture of 50 mM NaH(2)PO(4) (pH=2.5)-acetonitrile (30:70, v/v) as mobile phase with UV detection at 360 nm. The flow rate was set at 1.5 ml/min. The method was linear over the range of 0.05-5 microg/ml of gabapentin in plasma (r(2)>0.999). The within-day and between-day precision values were in the range of 2-5%. The limit of quantification of the method was 0.05 microg/ml. The method was successfully used to study the pharmacokinetics of gabapentin in healthy volunteers.     

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.     

Simultaneous determination of sulfamethoxazole and trimethoprim in biological fluids for high-throughput analysis: comparison of HPLC with ultraviolet and tandem mass spectrometric detection

The comparison of two methods based on online solid phase extraction-liquid chromatography with UV (SPE-LC-UV) or mass spectrometry detection (SPE-LC-MS/MS) for the simultaneous quantification of sulfamethoxazole (SMZ) and trimethoprim (TMP) is presented. The methods were validated and proved to be accurate. The analysis of standard samples for SMZ at concentrations of 0.5, 1.5, 25 and 50microg/mL demonstrated a relative standard deviation of less than 6% for both methods (n=18), while TMP samples at concentrations of 0.05, 0.15, 1.5 and 5.0microg/mL were analyzed with R.S.D. of less than 4% (n=18). The method with mass spectrometric detection was approximately six times more sensitive than the method with ultraviolet detection. The total run time for the SPE-LC-MS/MS was 2.5min per sample as opposed to 18.0min for the SPE-LC-UV method. The method with MS detection in comparison with UV detection proved to be more rugged and was successfully applied to pharmacokinetics studies.     

Determination of amiodarone and desethylamiodarone in horse plasma and urine by high-performance liquid chromatography combined with UV detection and electrospray ionization mass spectrometry

A rapid method for the quantification of amiodarone and desethylamiodarone in animal plasma using high-performance liquid chromatography combined with UV detection (HPLC-UV) is presented. The sample preparation includes a simple deproteinisation step with acetonitrile. In addition, a sensitive method for the quantification of amiodarone and desethylamiodarone in horse plasma and urine using high-performance liquid chromatography combined with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) is described. The sample preparation includes a solid-phase extraction (SPE) with a SCX column. Tamoxifen is used as an internal standard for both chromatographic methods. Chromatographic separation is achieved on an ODS Hypersil column using isocratic elution with 0.01% diethylamine and acetonitrile as mobile phase for the HPLC-UV method and with 0.1% formic acid and acetonitrile as mobile phase for the LC-MS/MS method. For the HPLC-UV method, good linearity was observed in the range 0-5 microg ml(-1), and in the range 0-1 microg ml(-1) for the LC-MS/MS method. The limit of quantification (LOQ) was set at 50 and 5 ng ml(-1) for the HPLC-UV method and the LC-MS/MS method, respectively. For the UV method, the limit of detection (LOD) was 15 and 10 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs of the LC-MS/MS method in plasma were much lower, i.e. 0.10 and 0.04 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The LODs obtained for the urine samples were 0.16 and 0.09 ng ml(-1) for amiodarone and desethylamiodarone, respectively. The methods were shown to be of use in horses. The rapid HPLC-UV method was used for therapeutic drug monitoring after amiodarone treatment, while the LC-MS/MS method showed its applicability for single dose pharmacokinetic studies.     

High performance liquid chromatographic determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE), a novel organoselenium compound, in dog plasma using pre-column derivatization and its application in pharmacokinetic study

A novel HPLC-UV method with pre-column derivatization by using 2-mercaptoethanol was established for determination of 1,2-[bis(1,2-benzisoselenazolone-3(2H)-ketone)]-ethane (BBSKE) in dog plasma. The derivatives were identified by mass spectrometry. The method had a good linear range of 0.05-2 microg/ml (r(2)=0.9995). The lower limit of quantification (LOQ) was 0.05 microg/ml. The precision and accuracy were less than 7%. After dosing of BBSKE (30 mg/kg, p.o. and 0.79 mg/kg, i.v.) in dogs, AUC(0-t) were 5.72+/-2.42 and 1.35+/-0.41 microg h/ml; t(1/2) were 4.6+/-2.1 and 1.7+/-0.6h, respectively. The method was successfully applied to the pharmacokinetic study in dogs.     

Quantification of rosuvastatin in human plasma by automated solid-phase extraction using tandem mass spectrometric detection

An assay employing automated solid-phase extraction (SPE) followed by high-performance liquid chromatography with positive ion TurboIonspray tandem mass spectrometry (LC-MS-MS) was developed and validated for the quantification of rosuvastatin (Crestor) in human plasma. Rosuvastatin is a hydroxy-methyl glutaryl coenzyme A reductase inhibitor currently under development by AstraZeneca. The standard curve range in human plasma was 0.1-30 ng/ml with a lower limit of quantification (LLOQ) verified at 0.1 ng/ml. Inaccuracy was less than 8% and imprecision less than +/-15% at all concentration levels. There was no interference from endogenous substances. The analyte was stable in human plasma following three freeze/thaw cycles and for up to 6 months following storage at both -20 and -70 degrees C. The assay was successfully applied to the analysis of rosuvastatin in human plasma samples derived from clinical trials, allowing the pharmacokinetics of the compound to be determined.     

Rapid determination of the synthetic pyrethroid insecticide, deltamethrin, in rat plasma and tissues by HPLC

Deltamethrin (DLM), [(S)-alpha-cyano-d-phenoxybenzyl-(1R,3R)-e-(2,2 dibromovinyl)-2,2-dimethylcyclo-propane-1-carboxylate], is a pyrethroid insecticide widely used in agriculture and households. There are several methods for analysis of DLM in biological fluids and tissues, but these methods are time consuming. They generally involve the extraction of DLM with lipid-soluble solvents such as n-pentane, n-hexane, diethylether or acetone, and subsequent evaporation of the solvent. A more rapid and sensitive high-performance liquid chromatography (HPLC) method to analyze DLM in plasma and tissues (liver, kidney, and brain) was developed and validated according to U.S. Food and Drug Administration (U.S. FDA) and International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. The limit of detection (S/N of 3/1) for DLM was 0.01 microg/ml for plasma, liver, kidney and brain. The method performances were shown to be selective for DLM and linear over the concentration range 0.01-20.0 microg/ml. For five replications of samples at 0.05, 0.1, 0.2, 1.5 and 4.0 microg/ml, intraday precision and accuracy values were in the range of 0.7-13.1% relative standard deviation (%R.S.D.) and 1.8-14.1%Error, respectively. Interday (n = 15) precision and accuracy values at 0.05, 0.1, 0.2, 1.5, and 4.0 microg/ml were in the range of 3.2-15.2% (%R.S.D.) and 3.7-14.8%Error, respectively. The absolute recoveries of DLM ranged from 93 to 103% for plasma, 95 to 114% for liver, 97 to 108% for kidney, and 95 to 108% for brain. This method can be quite useful for DLM pharmacokinetic and tissue distribution studies, for which multiple plasma and tissue samples have to be analyzed quickly with high reproducibility.     

Validated method for the determination of risperidone and 9-hydroxyrisperidone in human plasma by liquid chromatography-tandem mass spectrometry

Since the first entry of risperidone on to the market in the early 1990s, investigation of the pharmacokinetic behaviour of the compound for which the availability of a bioanalytical method was a condition sine qua non, has received considerable attention. Most of the published methods, however, did not reach the level of sensitivity and selectivity which can be obtained today since the evolution of liquid chromatography-tandem mass spectrometry (LC-MS-MS) towards a routine technique in the bioanalytical laboratory. Therefore, we developed and validated a new LC-MS-MS method for the determination of risperidone and its active metabolite 9-hydroxyrisperidone in human plasma. This paper describes in detail the bioanalytical procedure and summarizes the validation results obtained. In addition, it focuses on the pitfalls one might encounter when developing similar assays. Despite the particular physicochemical characteristics of risperidone and 9-hydroxyrisperidone, the LC-MS-MS method enabled the quantification of both compounds down to 0.1 ng/ml. The method uses a sample preparation step by solid-phase extraction at pH 6 using a mixed-mode phase. In a short chromatographic run, separation of 9-hydroxyrisperidone from the minor metabolite 7-hydroxyrisperidone is achieved. Detection takes place by (turbo)ionspray tandem mass spectrometry in the positive ion mode. The validated concentration range is from 0.100 to 250 ng/ml, using 500 microliter of sample, with accuracy (bias) and precision (coefficient of variation) being below 15%. Although new developments in equipment will allow us to further improve and speed up the method, the assay reported can be used as a routine method to support a wide range of pharmacokinetic studies.     

Stereoselective determination of benalaxyl in plasma by chiral high-performance liquid chromatography with diode array detector and application to pharmacokinetic study in rabbits

A chiral high-performance liquid chromatography method with diode array detector was developed and validated for stereoselective determination of benalaxyl (BX) in rabbit plasma. Good separation was achieved at 20 degrees C using cellulose tris-(3,5-dimethylphenylcarbamate) as chiral stationary phase, a mixture of n-hexane and 2-propanol (97:3) as mobile phase at a flow rate of 1.0 ml/min. The assay method was linear over a range of concentrations (0.25-25 microg/ml) in plasma and the mean recovery was greater than 90% for both enantiomers. The limits of quantification and detection for both enantiomers in plasma were 0.25 and 0.1 microg/ml, respectively. Intra- and interday relative standard deviations (RSDs) did not exceed 10% for three-tested concentrations. The method was successfully applied to pharmacokinetic studies of BX enantiomers in rabbits. The result suggested that the pharmacokinetics of BX enantiomers was stereoselective in rabbits.     

Determination of mildronate in human plasma and urine by liquid chromatography-tandem mass spectrometry

A sensitive and selective analytical method based on liquid chromatography-triple-quadrupole mass spectrometer has been developed to determine mildronate in human plasma and urine. The aim of this work was to find a valid method to study the pharmacokinetic profiles of mildronate in humans. Mildronate is a heart protection medicine, a carnitine's structural analogue, so levocarnitine was used as an internal standard for quantification. Under the electrospray ionization source positive ion mode, calibration curves with good linearities (r=0.9998 for plasma sample and r=0.9999 for urine sample) were obtained in the range of 1.0-20,000 ng ml(-1) for mildronate. The detection limit was 1 ng ml(-1). Recoveries were around 90% for the extraction from human plasma, and good precision and accuracy were achieved. This method is feasible for the evaluation of pharmacokinetic profiles of mildronate in humans, and to the best of our knowledge, this is the first report on LC-MS-MS analysis of mildronate in plasma and urine.     

Determination of 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA free acid) in rat plasma, urine and feces by liquid chromatography with UV and tandem mass spectrometric detection

BAPTA free acid was identified as the main metabolic product of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(actoxymethyl ester) (BAPTA-AM), a neuroprotective agent in cerebral ischemia, in rats. In this paper, liquid chromatography-ultraviolet (LC-UV) and mass spectrometry/mass spectrometry (LC-MS/MS) methods were employed for the determination of BAPTA free acid in rat urine and feces and rat plasma, respectively. By liquid-liquid extraction and LC-UV analysis, a limit of quantitation of 1000 ng/ml using 0.2 ml rat urine for extraction and 250 ng/ml using 1 ml rat fecal homogenate supernatant for extraction could be reached. The assay was linear in the range of 1000-50,000 ng/ml for rat urine and 250-10,000 ng/ml for rat fecal homogenate supernatant. Because the sensitivity of the LC-UV method was apparently insufficient for evaluating the pharmacokinetic profile of BAPTA in rat plasma, a LC-MS/MS method was subsequently developed for the analysis of BAPTA free acid. By protein precipitation and LC-MS/MS analysis, the limit of quantitation was 5 ng/ml using 0.1 ml rat plasma and the linear range was 5.0-500 ng/ml. Both methods were validated and can be used to support a thorough preclinical pharmacokinetic evaluation of BAPTA-AM liposome injection.     

Simple liquid chromatography-electrospray ionization mass spectrometry method for the routine determination of salmon calcitonin in serum

A simple liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) method was developed for the quantification of salmon calcitonin (sCT) in serum. Serum samples from rats and dogs were deproteinized and freeze-dried. The residue was then reconstituted with 57% acetonitrile in water containing 0.1% trifluoroacetic acid and 0.005% benzalkonium chloride. A 20-microl aliquot of the reconstituted solution was injected onto a polymer based RP-C18 column, The outlet was connected to an ion-trap mass spectrometer equipped with an ESI source, and spectra were recorded in a positive-ion, selected-ion monitoring mode. The limit of quantification of the method was 10 ng/ml. Biexponential curves were observed for the temporal serum concentration of sCT following intravenous administration of sCT to rats (100 microg/kg) and dogs (250 microg/kg), resulting in reasonable pharmacokinetic parameters. The present method appears applicable to routine analysis of serum sCT in pharmacokinetic studies with good selectivity, accuracy and precision.