Determination of a cyclic hexapeptide, a novel antifungal agent, in human plasma by high-performance liquid chromatography with ion spray and turbo ion spray tandem mass spectrometric detection

Methods for the determination of a semi-synthetic cyclic hexapeptide (I, MK-0991) in human plasma based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection using pneumatically assisted electrospray (ion spray, ISP) and turbo ion spray (TISP) interfaces were developed. Drug and internal standard (II, an isostere of I) were isolated from plasma by solid-phase extraction (SPE). The eluent from SPE was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The use of ISP, TISP and heated nebulizer (HN) interfaces as sample introduction systems were evaluated and showed that the heated nebulizer was not adequate for analysis due to thermal instability and/or adsorption of I and II to glass surfaces of the interface. Compounds I and II were chromatographed on a wide pore (300 Å), 150×4.6 mm C₈ analytical column, and the HPLC flow-rate of 1.2 ml/min was split 1:20 prior to introduction to the ISP or TISP interface of the mass spectrometric system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in selected reaction monitoring mode (SRM). The precursor→product ion combinations of m/z 1093.7→1033.6 and 1094.7→1033.6 were used to quantify I and II, respectively, after chromatographic separation of the analytes. The assay was validated in the concentration range of 10–1000 ng/ml using ISP, and 2.5–500 ng/ml of plasma using TISP with good precision and adequate accuracy. The effects of HPLC mobile-phase components on the ionization efficiency and sensitivity of detection in the positive ionization mode, the evaluation of the matrix effect, and limitations in sensitivity of detection of I due to the formation of multiply charged species are presented.     

Rapid determination of gemifloxacin in human plasma by high-performance liquid chromatography–tandem mass spectrometry

A method was developed for the determination of gemifloxacin (I) in human plasma using high-performance liquid chromatography–tandem mass spectrometry. Prior to analysis, the protein in plasma samples was precipitated with acetonitrile containing [¹³C²H₃] gemifloxacin (II) to act as an internal standard. The supernatant was injected onto a PLRP-S column without any further clean-up. The mass spectrometer was operated in positive ion mode, employing a heat assisted nebulisation, electrospray interface. Ions were detected in multiple reaction monitoring (MRM) mode. The assay requires 50 μl of plasma and is precise and accurate within the range 10–5000 ng/ml. The average within-run and between-run coefficients of variation were <11% at 10 ng/ml and greater concentrations. The average accuracy of validation standards was generally within ±7% of the nominal concentration. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can safely be stored for at least 6 months at −20°C. The method proved very robust and was successfully applied to the analysis of clinical samples from patients dosed with gemifloxacin.     

High-performance liquid chromatographic assay for the determination of the novel etoposide derivative dimethylaminoetoposide (NK611) and its metabolites in urine of cancer patients

A simple, reproducible and specific urine assay for the novel epipodophyllotoxin derivative dimethylaminoetoposide (NK611, I) its picro form (III), the N-demethyl metabolite (II) and its picro form (IV) is reported. The method involves the addition of Pr-NK611 as internal standard, chloroform extraction and HPLC separation on a Nova-Pak C₁₈ column with a mobile phase of acetonitrile-0.05 M KH₂PO₄ (pH 6.4) (23:77, v/v). UV detection was used with absorbance monitored at 205 nm and the limit of quantification was 100 ng/ml. The intra- and inter-day precisions were within the ranges 1.1–3.4% and 1.9–2.4% for all analytes and the accuracy was 101–107%. The extraction recovery was more than 88% for I, II and IV and more than 83% for III. The assay is applicable to the urinary monitoring of I–IV in clinical pharmacokinetic investigations.     

Determination of a novel selective inhibitor of type 1 5α-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay of human plasma for the determination of (5α,7β,16β)-16[(4-chlorophenyl)oxy]-4,7-dimethyl-4-aza-andronstan-3-one (I), a selective inhibitor of human type 1 5α-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid–liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor→product ion combinations of m/z 430→114 and 373→305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC–MS–MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.     

Screening of chlorpropamide in horse plasma by high-performance liquid chromatography with ultraviolet absorbance detection, and confirmation by gas chromatography–mass spectrometry

A chromatographic method was developed to detect and confirm the presence of chlorpropamide (I) in horse plasma samples, for antidoping control. The plasma sample (1 ml) was extracted with dichloromethane and screened by high-performance liquid chromatography, and confirmation of the drug's presence was accomplished by using gas chromatography–mass spectrometry (GC–MS). The limit of detection was found to be 3.5 ng/ml at a signal-to-noise ratio of three. Derivatization of I with N,O-bis-(trimethylsilyl)trifluoroacetamide with 1% trimethylchlorosilane allowed for highly stable, accurate and sensitive GC–MS analysis. Plasma samples collected after the administration of diabinese were positive for I (one–five days) in all samples analysed.     

Assay method for the perfluorooctyl bromide (perflubron) in rat blood by gas chromatography–mass spectrometry

This paper describes a GC–MS method (SIM mode) for the analysis of perfluorooctyl bromide (perflubron, I) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Following destruction of the emulsion by addition of ethanol, the analytical procedure involves a liquid–liquid extraction with 1,1,2-trichlorotrifluoroethane. The bis(F-butyl)ethene (II) was used as internal standard. Observed retention times were 3.22 min for I and 2.32 min for II. Two calibration curves were used; linear detection responses were obtained for concentrations ranging from 0.009 to 0.9 mg/ml and from 0.9 to 13.5 mg/ml. The extraction efficiency averaged 50% for I and 93% for II. Precision ranged from 0.7 to 14%, and accuracy was between 91 and 109%. The limit of quantification was 9 μg/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay method for use in pharmacokinetic studies.     

Quantification of perifosine, an alkylphosphocholine anti-tumour agent, in plasma by pneumatically assisted electrospray tandem mass spectrometry coupled with high-performance liquid chromatography

An HPLC assay with tandem mass spectrometric detection in the positive-ion Turbo-Ion-Spray^™ (TISP) mode for the fast and sensitive determination of perifosine ((I), D-21266) in human plasma was developed, utilising the structural analogue, miltefosine ((II), D-18506), as internal standard. Automated solid-phase extraction of diluted plasma samples, based on 250-μl plasma aliquots, at pH 6.5, allowed a reliable quantification of perifosine down to 4 ng/ml. Injection of 200 μl of plasma extracts onto a 100×3 mm normal-phase analytical column at a flow-rate of 0.5 ml/min provided retention-times of 2.4 and 2.1 min for perifosine (I) and the internal standard (II), respectively. The standard curves were linear from 4 to 2000 ng/ml using weighted linear regression analysis (1/Y²). The inter-assay and intra-assay accuracies for the calibration standards were within +0.9% and −0.2%, exhibiting precisions (C.V.) of ±6.5 and ±7.3%, respectively. Up to 100 unknowns may be analysed each 24 h per analyst.     

High-throughput, semi-automated determination of a cyclooxygenase II inhibitor in human plasma and urine using solid-phase extraction in the 96-well format and high-performance liquid chromatography with post-column photochemical derivatization-fluorescence detection

Compound I, 5-chloro-3-(4-methanesulfonylphenyl)-6′-methyl-[2,3′]bipyridinyl, has been found to be a specific inhibitor of the enzyme cyclooxygenase II (COX II). The anti-inflammatory properties of this compound are currently being investigated. HPLC assays for the determination of this analyte in human plasma and human urine have been developed. Isolation of I and the internal standard (II) was achieved by solid-phase extraction (SPE) in the 96-well format. A C₈ SPE plate was used for the extraction of the drug from human plasma (recovery >90%) while a mixed-mode (C₈/Cation) SPE plate was used to isolate the analytes from human urine (recovery approximately 71%). The analyte and internal standard were chromatographed on a Keystone Scientific Prism-RP^® guard column (20×4.6 mm) connected to a Prism-RP^® analytical column (150×4.6 mm), using a mobile phase consisting of 45% acetonitrile in 10 mM acetate buffer (pH=4); the analytes eluted at retention times of 5.2 and 6.9 min for I and II, respectively. Compounds I and II were found to form highly fluorescent products after exposure to UV light (254 nm). Thus, the analytes were detected by fluorescence (λ_ex=260 nm, λ_em=375 nm) following post-column photochemical derivatization. Eight point calibration curves over the concentration range of 5–500 ng/ml for human plasma and human urine yielded a linear response (R²>0.99) when a 1/y weighted linear regression model was employed. Based on the replicate analyses (n=5) of spiked standards, the within-day precision for both assays was better than 7% C.V. at all points on the calibration curve; within-day accuracy was within 5% of nominal at all standard concentrations. The between-run precision and accuracy of the assays, as calculated from the results of the analysis of quality control samples, was better than 8% C.V. and within 8% of nominal. I was found to be stable in human plasma and urine for at least 8 and 2 months, respectively. In addition, the human plasma assay was semi-automated in order to improve sample throughput by utilizing a Packard liquid handling system and a Tom-Tec Quadra 96 SPE system. The precision and accuracy of the semi-automated procedure were comparable to the manual procedure. Over 5000 clinical samples have been analyzed successfully using these methods.     

Automated high-performance liquid chromatography method for the determination of rosiglitazone in human plasma

A robust, fully automated assay procedure for the determination of rosiglitazone (I, BRL-49653) in human plasma has been developed. Plasma concentrations of I were determined using automated sequential trace enrichment of dialysates (ASTED) coupled to reversed-phase high-performance liquid chromatography. Sequential automated dialysis of human plasma samples was followed by concentration of the dialysate by trace enrichment on a C₁₈ cartridge. Drug and internal standard, SB-204882 (II) were eluted from the trace enrichment cartridge by mobile phase (0.01 M ammonium acetate, pH 8–acetonitrile, 65:35, v/v) onto the HPLC column (a Novapak C₁₈, 4 μm, 100×5 mm radial compression cartridge) protected by a Guard-Pak C₁₈ cartridge. The compounds were detected by fluorescence detection, using an excitation wavelength of 247 nm, and emission wavelength of 367 nm. The lower limit of quantitation of the method was 3 ng/ml (200 μl aliquot) with linearity demonstrated up to 100 ng/ml. Within- and between-run precision and accuracy of determination were better than 10% across the calibration range. There was no evidence of instability of I in human plasma following three complete freeze–thaw cycles and samples can be safely stored for at least 7 months at −20°C. This method has been successfully utilised to provide pharmacokinetic data throughout the clinical development of rosiglitazone.     

Determination of a thermally labile metabolite of a novel growth hormone secretagogue in human and dog plasma by liquid chromatography with ion spray tandem mass spectrometric detection

A sensitive and selective assay for the determination of N-{1(R)-[(1,2-dihydro-1-methylsulfonylspiro[3H-indole-3,4′-piperidin]-1′-yl)carbonyl]-2-(phenylmethoxy)-ethyl}-2-hydroxyamino-2-methylpropanamide (I), a hydroxyl amine metabolite of a novel growth hormone secretagouge (II) has been developed utilizing high-performance liquid chromatography with ion spray tandem mass spectrometric detection (HPLC–MS–MS). The analyte and an internal standard (III) were isolated from the basified biological matrix using a liquid–liquid extraction with methyl tert.-butyl ether (MTBE). The organic extract was evaporated to dryness at room temperature. The residue was reconstituted in the mobile phase and injected into the HPLC–MS–MS system. Multiple reaction monitoring using the precursor→product ion combinations of m/z 545→267 and 543 →267 was used to quantify I and III, respectively, after chromatographic separation under isocratic conditions. The assay was validated in the concentration range of 0.5 to 500 ng/0.1 ml in both human and dog plasma. The precision of the assay, expressed as relative standard deviation, was less than 10% over the entire concentration range with the exception of the low concentration of 0.5 ng/0.1 ml which was 14.0% for human plasma. The HPLC–MS–MS method provided sufficient sensitivity to completely map the pharmacokinetic time course of I following a single 5 mg dose of II to human subjects and a 0.5 mg/kg dose to beagle dogs.     

Rapid high-performance liquid chromatographic determination of docetaxel (Taxotere) in plasma using liquid–liquid extraction

A new rapid and sensitive high-performance liquid chromatographic method for analysis of docetaxel (Taxotere) in human plasma was developed and validated. After adding an internal standard (paclitaxel, Taxol), plasma was extracted following a simple liquid–liquid extraction with diethyl ether. Extraction efficiency averaged 95% for docetaxel. Separation was performed using a Nucleosil (C₁₈) 5 μm column, monitored at 227 nm. The isocratic mobile phase consisted of acetonitrile–acetate buffer, pH 5–tetrahydrofuran (45:50:5, v/v) pumped at a flow-rate of 1.8 ml/min. The limit of quantification for docetaxel in plasma was 12.5 ng/ml. Retention times for docetaxel and paclitaxel were 7.7 and 9 min, respectively. Standard curves were linear over a range of 25–1000 ng/ml. This new method is rapid since it does not require time-consuming extraction procedures, or complex chromatographic conditions. This rapidity, along with the lack of chromatographic interferences with various other drugs likely to be administered to the cancer patients (pain killers, corticoids, antiemetics drugs) make this method suitable for daily routine analysis of Taxotere, a major anticancer drug extensively used in clinical oncology.     

Determination of clindamycin in human plasma by liquid chromatography–electrospray tandem mass spectrometry: application to the bioequivalence study of clindamycin

A simple, sensitive and specific liquid chromatography–electrospray tandem mass spectrometry (LC–MS–MS) method for the determination of clindamycin (I) was developed. Both I and verapamil (II, internal standard) were analyzed using a C₁₈ column with a mobile phase of 80% acetonitrile–0.01% trifluoroacetic acid. Column eluents were monitored by electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to daughter combinations of m/z 425→126 and 455→165 was used to quantitate I. A limit of quantitation of 0.0500 μg/ml was found. The assay exhibited a linear dynamic range of 0.0500–20.0 μg/ml and gave a correlation coefficient (r²) of 0.998 or better. The chromatographic run time was approximately 2 min. The intra-batch precision and accuracy of the quality controls (QCs, 0.0500, 0.150, 1.50, 15.0 and 20.0 μg/ml) were characterized by coefficients of variation (CVs) of 5.13 to 13.7% and relative errors (REs) of −4.34 to 4.58%, respectively. The inter-batch precision and accuracy of the QCs were characterized by CVs of 4.35 to 8.32% and REs of −10.8 to −4.17%, respectively. The method has successfully been applied to the analysis of samples taken up to 12 h after oral administration of 300 mg of I in healthy volunteers.     

Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography–tandem mass spectrometry

A rapid, selective and sensitive HPLC–tandem mass spectrometry method was developed and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal standard diphenhydramine were extracted from plasma samples by liquid–liquid extraction, and chromatographed on a C₁₈ column. The mobile phase consisted of acetonitrile–water–formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml/min. Detection was performed on a triple quadrupole tandem mass spectrometer by selected reaction monitoring (SRM) mode via atmospheric pressure chemical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The linear calibration curves were obtained in the concentration range of 10.0–1500.0 ng/ml for flupirtine and 2.0–300.0 ng/ml for D-13223. The intra- and inter-run precision (RSD), calculated from quality control (QC) samples was less than 7.2% for flupirtine and D-13223. The accuracy as determined from QC samples was less than 5% for the analytes. The overall extraction recoveries of flupirtine and D-13223 were determined to be about 66% and 78% on average, respectively. The method was applied for the evaluation of the pharmacokinetics of flupirtine and active metabolite D-13223 in volunteers following peroral administration.     

Rapid determination of citalopram in human plasma by high-performance liquid chromatography

A rapid high-performance liquid chromatographic method for the quantitation of citalopram in human plasma is presented. The sample preparation involved liquid–liquid extraction of citalopram with hexane–isoamyl alcohol (98:2 v/v) and back-extraction of the drug to 0.02 M hydrochloric acid. Liquid chromatography was performed on a cyano column (45×4.6 mm, 5 μm particles), the mobile phase consisted of an acetonitrile–phosphate buffer, pH 6.0 (50:50, v/v). The run time was 2.6 min. The fluorimetric detector was set at an excitation wavelength of 236 nm and an emission wavelength of 306 nm. Verapamil was used as the internal standard. The limit of quantitation was 0.96 ng/ml using 1 ml of plasma. Within- and between-day precision expressed by relative standard deviation was less than 7% and inaccuracy did not exceed 6%. The assay was applied to the analysis of samples from a pharmacokinetic study.     

A high-performance liquid chromatography assay with ultraviolet detection for olanzapine in human plasma and urine

Olanzapine is a commonly used atypical antipsychotic medication for which therapeutic drug monitoring has been proposed as clinically useful. A sensitive method was developed for the determination of olanzapine concentrations in plasma and urine by high-performance liquid chromatography with low-wavelength ultraviolet absorption detection (214 nm). A single-step liquid–liquid extraction procedure using heptane-iso-amyl alcohol (97.5:2.5 v/v) was employed to recover olanzapine and the internal standard (a 2-ethylated olanzapine derivative) from the biological matrices which were adjusted to pH 10 with 1 M carbonate buffer. Detector response was linear from 1–5000 ng (r²>0.98). The limit of detection of the assay (signal:noise=3:1) and the lower limit of quantitation were 0.75 ng and 1 ng/ml of olanzapine, respectively. Interday variation for olanzapine 50 ng/ml in plasma and urine was 5.2% and 7.1% (n=5), respectively, and 9.5 and 12.3% at 1 ng/ml (n=5). Intraday variation for olanzapine 50 ng/ml in plasma and urine was 8.1% and 9.6% (n=15), respectively, and 14.2 and 17.1% at 1 ng/ml (n=15). The recoveries of olanzapine (50 ng/ml) and the internal standard were 83±6 and 92±6% in plasma, respectively, and 79±7 and 89±7% in urine, respectively. Accuracy was 96% and 93% at 50 and 1 ng/ml, respectively. The applicability of the assay was demonstrated by determining plasma concentrations of olanzapine in a healthy male volunteer for 48 h following a single oral dose of 5 mg olanzapine. This method is suitable for studying olanzapine disposition in single or multiple-dose pharmacokinetic studies.     

Determination of nadolol diastereomers in dog plasma using chiral derivatization and reversed-phase high-performance liquid chromatography with fluorescence detection

A stereospecific high-performance liquid chromatographic method has been developed for the determination of four diastereomers of nadolol in plasma. After the nadolol diastereomers were extracted from plasma using an Extrelut-1 solid-phase extraction cartridge, they were derivatized with (R)-(−)-1-(1-naphthyl)ethylisocyanate to form urea derivatives. These derivatives were then separated on a YMC-AM-303 ODS column using water—acetonitrile (60:40, v/v). The calibration curves of (SR)-, (RS)-, (SS)- and (RR)-nadolol were linear over the range 2.5–200 ng/ml, and the correlation coefficient (r) of the curves were higher than 0.9991 for each diastereomer. The limit of quantification was 2.5 ng/ml for each diastereomer in plasma. This method was used for a pharmacokinetic study in four dogs after oral administration of nadolol (1 mg/kg). The plasma concentrations of nadolol diastereomers showed no significant differences in C_max, T_max or AUC values. The assay appears to be readily applicable to the study of diastereoselective nadolol pharmacokinetics in animals and humans.     

Determination of vinorelbine in rabbit plasma by high-performance liquid chromatography with coulometric detection

A high-performance liquid chromatographic method was developed for the determination in plasma (400-μl sample) of a vinca alkaloid, vinorelbine. The analysis was performed by using an octadecylsilane column and heptanesulfonic acid as ion-pairing agent. This method used a new internal standard, teniposide, that permitted a good compromise between sensitivity and retention times (10.6 and 15.5 min for teniposide and vinorelbine, respectively). After a liquid-liquid extraction with diethyl ether, the extracts were injected into a reversed-phase system. The extraction efficiency was approximately 80% for both vinorelbine and the internal standard. The mobile phase was phosphate buffer (pH 3)-acetonitrile-methanol (50:30:20, v/v/v). Using coulometric detection, the limit of detection in plasma (400 μl) was 1 ng.ml. The intra-assay coefficients of variation were 10.95, 3.80 and 5.71% for 5, 500 and 1000 ng/ml, respectively, and the inter-assay coefficients of variation were 20.14, 14.27 and 10.67% for 5, 500 and 1000 ng/ml, respectively. A linear response was observed for the plasma calibration graph in the ranges 2.5–50 and 50–1000 ng/ml. This method was used to follow the time course of the concentration of vinorelbine in rabbit plasma after a single intravenous dose of vinorelbine (30 mg/m²) and seems to be suitable for studying the pharmacokinetics of vinorelbine in rabbit.     

Determination of a new antimalarial drug, benflumetol, in blood plasma by high-performance liquid chromatography

A rapid and selective high-performance liquid chromatographic assay for determination of a new antimalarial drug (benflumetol, BFL) is described. After extraction with hexane-diethyl ether (70:30, v/v) from plasma, BFL was analysed using a C₁₈ Partisil 10 ODS-3 reversed-phase stainless steel column and a mobile phase of acetonitrile-0.1 M ammonium acetate (90:10, v/v) adjusted to pH 4.9 with ultraviolet detection at 335 nm. The mean recovery of BFL over a concentration range of 50–400 ng/ml was 96.8±5.2%. The within-day and day-to-day coefficients of variation were 1.8–4.0 and 1.8–4.2%, respectively. The minimum detectable concentration in plasma for BFL was 5 ng/ml with a C.V. of less than 10%. This method was found to be suitable for clinical pharmacokinetic studies.     

High-performance liquid chromatographic determination of vinorelbine in human plasma and blood: application to a pharmacokinetic study

A sensitive and specific high-performance liquid chromatographic method with fluorescence detection (excitation wavelength: 280 nm; emission wavelength: 360 nm) was developed and validated for the determination of vinorelbine in plasma and blood samples. The sample pretreatment procedure involved two liquid–liquid extraction steps. Vinblastine served as the internal standard. The system uses a Spherisorb cyano analytical column (250×4.6 mm I.D.) packed with 5 μm diameter particles as the stationary phase and a mobile phase of acetonitrile–80 mM ammonium acetate (50:50, v/v) adjusted to pH 2.5 with hydrochloric acid. The assay showed linearity from 1 to 100 ng/ml in plasma and from 2.5 to 100 ng/ml in blood. The limits of quantitation were 1 ng/ml and 2.5 ng/ml, respectively. Precision expressed as RSD was in the range 3.9 to 20% (limit of quantitation). Accuracy ranged from 92 to 120%. Extraction recoveries from plasma and blood averaged 101 and 75%, respectively. This method was used to follow the time course of the concentration of vinorelbine in human plasma and blood samples after a 10-min infusion period of 20 mg/m² of this drug in patients with metastatic cancer.     

Determination of trans-resveratrol in human plasma by high-performance liquid chromatography

The first method using high-performance liquid chromatography (HPLC) has been developed for the determination of trans-resveratrol in human plasma. The method involves a liquid–liquid extraction followed by reversed-phase HPLC with UV detection. The detection limit of trans-resveratrol in human plasma was 5.0 ng/ml. Standard curves are linear over the concentration range of 5.0–5000.0 ng/ml. Intra-assay variability ranged from 1.9 to 3.7% and inter-assay variability ranged from 2.5 to 4.0% at the concentration range of 15.0–4000.0 ng/ml.