Optimization and clinical assessment of a radioreceptor assay for thyrotropin-binding inhibitor immunoglobulins

We tried to improve the sensitivity of a radioreceptor assay for thyrotropin-binding inhibitor immunoglobulins (TBII) by modifying assay conditions. About a twofold increase in sensitivity without a loss of reproducibility was obtained by prolonging the incubation of the receptors with test serum from 15 to 120 min before the addition of 125I-labeled thyrotropin. In 20 untreated, 49 treated patients with Graves' disease and 19 patients with euthyroid Graves' disease, TBII activities obtained using 120 min preincubation were significantly higher than those obtained using 15 min preincubation (p less than 0.005). No significant increase in TBII activities was observed in the presence of sera from patients with primary hypothyroidism (n = 17), simple goiter (n = 7), adenomatous goiter (n = 11), thyroid adenoma (n = 11) or cancer (n = 12). TBII were detectable in 15 (47%) of 32 triiodothyronine-nonsuppressible Graves' patients who were receiving maintenance antithyroid drug therapy using 120 min preincubation, while they were positive in only 6 patients (19%) using 15 min preincubation. The assay using a longer preincubation period was found to be sensitive, specific and useful for diagnosis and follow-up of Graves' disease.     

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.     

Quantification of CPT13 in rat plasma using LC-MS/MS for a pharmacokinetic study

A new, simple, sensitive and specific reversed-phase high performance liquid chromatographic (HPLC) method using tandem mass spectrometry detection was initially developed and validated for the analysis of 10-(2-pyrazolyl-ethoxy)-(20S)-camptothecin (CPT13) in rat plasma. Pretreatment of the sample obtained from plasma involved a single protein precipitation step with using acetonitrile containing 0.1% formic acid. An aliquot of 20 μl was injected into a C-18 column. The chromatographic separation was achieved using the mobile phase consisting of acetonitrile:water (35:65) at a flow rate of 1.0 mL/min. The total run time for each sample was 10 min, and camptothecin (CPT, IS) and CPT13 were well separated with retention times of 5.1 min and 5.6 min, respectively. Detection was performed using a triple quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) source. The calibration curve was linear (r2 = 0.9998) over the concentration range of 1-1000 ng/mL, with a LLOQ of 1 ng/mL for CPT13. The inter- and intra-day precision (%R.S.D.) were <2.58% and 6.28%, respectively, and the accuracies (%) were within the range of 97.34-110.67%. CPT13 in rat plasma was stable when stored at -20 °C or 4 °C for three freeze-thaw cycles, The method was employed for the first time during pharmacokinetic studies of CPT13 in rats following a single intravenous dose (0.1 mg/kg) and three different oral doses (50 mg/kg, 30 mg/kg, and 10 mg/kg). This fully validated method was successfully applied to a pharmacokinetic study of CPT13 in rats.     

Rapid Immunocytochemistry with Simple Heat-Induced Antigen Retrieval Technique for Improvement in the Quality of Cytological Diagnosis

Rapid immunocytochemistry (ICC) can improve the accuracy of intraoperative cytological diagnoses; however, it is usually applied without heat-induced antigen retrieval (HIAR). We established a HIAR method for rapid ICC and evaluated its efficacy and reliability. Rapidly fixed smear samples were immunostained using 35 antibodies. We compared the results of HIAR by boiling in a pot or heating in an electric kettle. The smears were incubated for 3 min with each primary antibody and immuno-enzyme polymer reagent, and for 1 min with diaminobenzidine solution. HIAR for 1 min using the kettle method yielded the best cellular integrity. For 32 out of the 35 antibodies, results achieved using rapid ICC within 11 min were comparable to that achieved using standard ICC. HIAR was essential for 13 antibodies. For two of the antibodies, HIAR was not required when standard ICC was applied, but consistent staining with rapid ICC was obtained only with HIAR. In conclusion, we established a rapid ICC procedure using a simple HIAR method, which allowed efficient immunostaining of a panel of antigens, including nuclear antigens, within only 11 min. The combined use of this rapid ICC technique with other staining techniques could be useful for improving intraoperative cytological diagnoses.     

Metabolic clearance rate of rat prolactin in preweanling rat pups

The metabolic clearance rate (MCR) of immunoreactive rat prolactin (rPRL) was determined in urethane-anaesthetized 14 to 16-day-old rat pups using the constant infusion to equilibrium method. Two different doses of rPRL were used, 329 and 472 ng/min each at a volume of 0.0025 ml/min for 30 min. The MCR was 0.216 ml/min following infusion of 329 ng PRL/min dose and 0.337 ml/min following infusion of the 472-ng/min dose. When calculated on the basis of metabolic body weight the MCR of rPRL in rat pups was comparable to that of adult rats.     

A high-performance liquid chromatography with electrochemical detection for the determination of total hypericin in extracts of St. John's Wort

An HPLC method for the quantitation of hypericin using a new and sensitive amperometric detection is presented. Hypericin was eluted isocratically using a mobile phase consisting of ammonium acetate, methanol and acetonitrile. The oxidation was carried out with a glassy carbon electrode at a potential of + 1.1 V vs. an Ag-AgCl-KCl reference electrode. Under the conditions described, hypericin was separated at a retention time (Rt) of 12 min. Linearity was obtained over the range 0.035-1.30 microg/mL (r = 0.9994). The limit of detection was determined to be 0.010 ng on-column for hypericin. The method was applied to the determination of total hypericin (hypericin, pseudohypericin, protohypericin and protopseudohypericin) in extracts of St. John's wort using hypericin as an external standard. The protoforms were converted into hypericin and pseudohypericin by subjecting the sample to artificial light prior to chromatographic analysis. For the evaluation of total hypericin, the peak areas of pseudohypericin (Rt 3.7 min) and hypericin (Rt 12.0 min) were combined. The relative standard deviation in analysing samples containing Hypericum ranged from 2.5 to 5.4%.     

The validation of a bioanalytical method for the determination of clopidogrel in human plasma

A fast, sensitive and specific LC-MS/MS bioanalytical method for the determination of unchanged clopidogrel in human plasma has been developed and validated over the range of 10-12,000 pg mL(-1) (r2 0.9993) by the Contract Research group at HFL. Samples (0.3 mL) were buffered (pH 6.8), extracted using diethyl ether and 10 microL of the sample extract was injected onto the LC-MS/MS system. Analysis was performed using a C8 column (temperature controlled to 50 degrees C) by gradient elution at a flow rate of 0.9 mL min(-1) over a 3 min run time. Retention times of 1.61 and 1.59 min were observed for clopidogrel and 2H3-clopidogrel (I.S.), respectively. Detection was achieved using a Sciex API 4000, triple quadrupole mass spectrometer, in positive TurboIonspray (electrospray) ionisation mode. Ion transitions were monitored using MRM (multiple reaction monitoring) for clopidogrel (m/z 322-212) and for 2H3-clopidogrel (m/z 327-217). This validated method was used to support a pharmacokinetic study in healthy volunteers.     

Determination of isoflavones in soybean food and human urine using liquid chromatography with electrochemical detection

A highly sensitive high-performance liquid chromatographic method with electrochemical detection (HPLC-ED) was developed for the determination of isoflavones. Electrochemical behaviour of daidzein and genistein was studied on carbon paste electrode (CPE) by adsorptive transfer stripping square wave voltammetry. The obtained electrochemical results were used for the development of HPLC-ED method. Furthermore, isoflavones were separated on an Atlantis dC18 column using a mobile phase consisting of acetonitrile (solvent A) and 0.15M acetate buffer of pH 5.5 (solvent B) at a flow rate 0.4 mL/min. A linear gradient profile (solvent B) was at 0-2 min 87%; 22 min 60%; 27 min 50%; 31 min 45%; 47 min 87%. Full scan of multi-channel coulometric detection was tested and optimal potential at 450 mV was chosen for our purposes. Calibration curves were linear (daidzein R(2) = 0.9993 and genistein R(2) = 0.9987). The detection limit for daidzein/genistein was 480/394 pg/mL (1.8/1.5 nM) and per column 2.4/1.9 pg. Isoflavones extracted from soybean products (farina, meat, milk) by the accelerated solvent extraction (ASE) procedure and isoflavones present in human urine were determined by the HPLC-ED method.     

Purification and characterization of carbonic anhydrase from bovine stomach and effects of some known inhibitors on enzyme activity

Carbonic anhydrase (CA) was purified from four different cell localisation (outer peripheral, cytosolic, inner peripheral and integral) in bovine stomach using affinity chromatography with Sepharose-4B-L-tyrosine sulphanilamide. During the purification steps, the activity of the enzyme was measured using p-nitrophenyl acetate at pH 7.4. Optimum pH and optimum temperature values for all CA samples were determined, and their K(m) and V(max) values for the same substrate by Lineweaver-Burk graphics. The extent of purification for all CA localizations was controlled by SDS-PAGE. The K(m) values at optimum pH and 20 degrees C were 0.625 mM, 0.541 mM, 0.785 mM and 0.862 mM with p-nitro phenyl acetate, for all CA localizations. The respective V(max) values at optimum pH and 20 degrees C were 0.875 micromol/L min, 0.186 micromol/L min, 0.214 micromol/L min and 0.253 micromol/L min with the same substrate. The K(i) and I50 values for the inhibitors sulphanilamide, KSCN, NaN3 and acetazolamide were determined for all the CA localizations.     

Water movement in tendon in response to a repeated static tensile load using one-dimensional magnetic resonance imaging

Rabbit Achilles tendons (N = 8) were subjected to tensile loading while internal water movements were followed using NMR. The distribution of the internal water in tendons was measured using a one-dimensional proton-density map that was collected along a radial line oriented transverse to the tendon's long axis. The proton density map was created from fits to T2 relaxation data. The experimental design included two cycles of loading (7.5 N tensile load) and relaxation. The first load application was for 42.67 min: unloaded for 21.33 min, reloaded for 21.33 min, and then unloaded for 21.33 min. Water was redistributed in a time-dependent fashion upon loading: proton density decreased in the core region and increased in the rim region. In addition there was evidence that tensile loading caused water to become NMR visible. In separate, parallel experiments, we studied the mechanical behavior of tendons using identical conditions of uniaxial loading (N = 7). The time constants of water movements were very different from the time constants of mechanical relaxation, indicating that water redistribution is not the sole determining factor of mechanical behavior.     

Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore

Published values for sperm membrane water permeability (L(p)) obtained using a time-to-lysis methodology have produced anomalous results when used to model optimal cooling rates for cryopreservation of spermatozoa. As the lysis method is dependent on potentially questionable assumptions, we describe an alternative method for measuring sperm L(p). Spermatozoa were exposed to hypo- and hyperosmotic conditions using a stopped-flow apparatus and the time course of resulting volume changes was measured using concentration-dependent self-quenching of the entrapped fluorophore, carboxyfluorescein (CF). L(p) was measured for boar, rabbit, and ram spermatozoa using a range of osmotic stresses (+/-50-100 mOsm). Values for exosmotic and endosmotic flow showed no evidence of rectification. Mean L(p) values were 0.84 microm/min/atm (boar), 0.28 microm/min/atm (rabbit), and 2.79 microm/min/atm (ram). These values are lower than the lysis method estimates, with the ram value reduced by approximately two-thirds using the current methodology. The value for boar spermatozoa showed good agreement with published values obtained using an electronic cell-sizing technique. Substitution of the revised values for L(p) into the model for optimal cooling rates brings the calculated optimal rate closer to the lower empirically observed value but does not fully account for the previously reported discrepancies.     

The Variability in Circadian Phase and Amplitude Estimates Derived from Sequential Constant Routines

Both the constant routine (CR) and the dim light melatonin onset have been suggested as reliable methods to determine circadian phase from a single circadian cycle. However, both techniques lack published studies quantifying the intercycle variability in their phase resolution. To address this question eight healthy male subjects participated in two CRs, 7 days apart. Circadian phase was determined using 3-min samples of core body temperature and two hourly urinary sulphatoxy melatonin excretion rates. Phase and amplitude were estimated using simple (24 h) and complex (24 + 12 h) cosinor models of temperature data and the onset, offset, and a distance-weighted-least-squares (DWLS) fitted acrophase for the melatonin metabolite. The variability in phase estimates was measured using the mean absolute difference between successive CRs. Using the simple 24 h model of temperature data, the mean absolute phase difference was 51 min (SD = 35 min). Using the complex model, the mean absolute phase difference was 62 min (SD = 35 min). Using the DWLS fitted acrophase for the melatonin metabolite, the mean absolute phase difference between CR1 and CR2 was 40 min (SD = 26 min). The results indicate that for CRs a week apart, the mean absolute difference in an individual's phase estimate can vary by 40-60 min depending on the choice of dependent measure and analytic technique. In contrast to the intraindi-vidual variability, the group results showed considerably less variability. The mean algebraic difference between CRs, using temperature- or melatonin-derived estimates, was less than 5 min, and well within the range of normal measurement error.     

HPLC-atmospheric pressure chemical ionization mass spectrometric method for enantioselective determination of R,S-propranolol and R,S-hyoscyamine in human plasma

A method for the simultaneous determination of R- and S-propranolol and R- and S-hyoscyamine in human plasma was developed, validated and applied to the analysis of samples from a clinical study. Sample preparation was performed by solid-phase extraction of 2 ml of human plasma using Oasis MCX cartridges and the enantioselective separations were achieved using a Chirobiotic V chiral stationary phase. The chromatography was carried out using gradient elution with a mobile phase composed of methanol:acetic acid:triethylamine which was varied from 100:0.05:0.04 to 100:0.05:0.1 (v/v/v) over 30 min and delivered at a flow rate 1 ml/min. The internal standard was R,S-propranolol-d7 and the analytes were quantified using a single quadrupole mass spectrometer employing APCI interface operated in the positive ion mode with single ion monitoring. The enantioselective separation factors, alpha, were 1.15 and 1.07 for S- and R-propranolol and R- and S-hyoscyamine, respectively. The standard curves were linear for all target compounds with coefficients of determination (r2) ranging from 0.9977 to 0.9999. The intra- and inter-day precision and accuracy were 相似文献   

Development and validation of a high-performance liquid chromatography-mass spectroscopy assay for determination of artesunate and dihydroartemisinin in human plasma

A sensitive method has been developed and validated for the determination of artesunate and its active metabolite dihydroartemisinin (DHA) in human plasma using artemisinin as an internal standard. Solid phase extraction (SPE) using Oasis HLB extraction cartridges was used for sample preparation and analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. Positive ions were measured using extracted ion chromatogram mode. The extracted ion for artesunate, alpha- and beta-DHA was m/z 221 and for artemisinin was m/z 283. Chromatography was carried out using a Synergi Max-RP, 4 mu, 75 mm x 4.6 mm column using glacial acetic acid 0.1%, acetonitrile and methanol mixture (38:46.5:15.5) as a mobile phase delivered at a flow rate of 0.5 mL/min. The retention times of artesunate, alpha- and beta-DHA and artemisinin were 17.4, 11.8, 18.7 and 13.4 min, respectively, with a total run time of 21 min. The assay was linear over the range 1-3000 ng/mL for artesunate and DHA. The analysis of quality control samples for artesunate 50, 300, 1300 and 2600 ng/mL demonstrated excellent precision with relative standard deviation of 14.3, 11.3, 7.5 and 12.1%, respectively (n=5). Recoveries at concentration of 50, 300, 1300 and 2600 ng/mL were 75, 94.5, 74.3 and 75.5%, respectively; similar results were obtained for precision and recovery of DHA. This liquid chromatography-mass spectroscopy (LC-MS) method for the determination of artesunate and DHA in human plasma has superior specification for sensitivity, sample throughput and robustness than previous methods and can reliably quantitate concentrations of both (artesunate and DHA) compounds as low as 1 ng/mL.     

Immobilisation of free-living Weddell seals Leptonychotes weddellii using midazolam and isoflurane

Eleven lactating female Weddell seals were immobilised using inhaled isoflurane and oxygen, having initially been sedated using an intramuscular injection of midazolam. The seals were selected from colonies in Long Fjord, East Antarctica. Isoflurane was delivered using a precision, out-of-circle vaporiser in a portable, heated, semi-closed circle system anaesthetic machine. Induction time (time from injection of midazolam to detected maximal effect of midazolam) ranged from 12 min to 29 min. The maximal effect of midazolam was assessed as being either moderate sedation (n=9) or heavy sedation (n=2), and the maximal effect of inhaled isoflurane and oxygen was assessed as being light anaesthesia (n=11). The level of chemical restraint achieved using this combination allowed attachment of heart rate monitoring units and collection of biological samples. Recovery time ranged from 1 min to 11 min. The anaesthetic regime proved a practical, safe and reliable method for the immobilisation of lactating Weddell seals under conditions of low environmental temperature.     

Kinetic studies on the adsorption of Cd2+, Cu2+ and Zn2+ ions from aqueous solutions by cassava (Manihot sculenta Cranz) tuber bark waste

The kinetics of Cd2+, Cu2+ and Zn2+ adsorption onto pure and thioglycolic acid treated cassava tuber bark wastes (CTBW) were investigated using a batch sorption technique at 30 degrees C. Kinetic data suggested that the adsorption process was exothermic, the rate limiting sorption step was physisorption and adsorption rates could be best described by a pseudo-second order model. Rate coefficients were determined to range between 1.39x10(-2)min(-1) and 5.94x10(-2)min(-1), 1.46x10(-3)min(-1) and 5.76x10(-3)min(-1) and 0.69x10(-3)min(-1) and 5.8x10(-3)min(-1) for Cd2+, Cu2+ and Zn2+, respectively. The results from these studies indicated that the sorption process is fast and stable. The adsorption equilibria were evaluated using the Langmuir equation and the monolayer sorption capacity was found to range between 5.88-26.3mg/g, 33.3-90.9 mg/g and 22.2-83.3mg/g for Cd2+, Cu2+ and Zn2+, respectively. Negative values of DeltaG(ads)(0) indicated that the adsorption process was spontaneous and exothermic in nature.     

Phenotype of CYP2C19 and CYP3A4 by determination of omeprazole and its two main metabolites in plasma using liquid chromatography with liquid-liquid extraction

We present a new simple and reliable HPLC method for measuring omeprazole and its two main metabolites in plasma. This can be used for studying CYP2C19 and CYP3A4 genetic polymorphisms using omeprazole as the probe drug. Omeprazole, hydroxyomeprazole and omeprazole sulfone were extracted from plasma samples with phosphate buffer and dichloromethane-ether (95:5). HPLC separation was achieved using an Ultrasphere ODS C(18) (Beckman) column. The mobile phase was acetonitrile-phosphate buffer (24:76, pH 8), containing nonylamine at 0.015%. Retention times were 9.5 min for omeprazole, 3.25 min for hydroxyomeprazole, 7.4 min for omeprazole sulfone and 6.27 min for internal standard (phenacetine). Detection (UV at 302 nm) of analytes was linear in the range from 96 to 864 ng/ml. This is useful for calculating metabolic index for CYP2C19 and CYP3A4 in adults and children. This method is stable, reproducible, improves resolution and has practical advantages such as low cost.     

Development and validation of a highly sensitive and robust LC-MS/MS with electrospray ionization method for simultaneous quantitation of itraconazole and hydroxyitraconazole in human plasma: application to a bioequivalence study

A highly sensitive and specific LC-MS/MS method has been developed for simultaneous estimation of itraconazole (ITZ) and hydroxyitraconazole (OH-ITZ) with 500 microL of human plasma using fluconazole as an internal standard (IS). The API-4000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Solid phase extraction process was used to extract ITZ, OH-ITZ and IS from human plasma. The total run time was 3.0 min and the elution of ITZ, OH-ITZ and IS occurred at 2.08 min, 1.85 min and 1.29 min, respectively; this was achieved with a mobile phase consisting of 0.2% (v/v) ammonia solution:acetonitrile (20:80, v/v) at a flow rate of 0.50 mL/min on a HyPurity C(18) (50 mm x 4.6 mm, 5 microm) column. The developed method was validated in human plasma with a lower limit of quantitation of 0.50 ng/mL for both ITZ and OH-ITZ. A linear response function was established for the range of concentrations 0.5-263 ng/mL (r>0.998) for both ITZ and OH-ITZ. The intra- and inter-day precision values for ITZ and OH-ITZ met the acceptance as per FDA guidelines. ITZ and OH-ITZ were stable in the battery of stability studies, viz., bench-top, auto-sampler, dry extract and freeze/thaw cycles. The developed assay method was applied to an oral bioequivalence study in humans.     

Capillary electrophoresis-based nanoscale assays for monitoring ecto-5'-nucleotidase activity and inhibition in preparations of recombinant enzyme and melanoma cell membranes

Powerful capillary electrophoresis (CE) methods were developed for monitoring the reaction of ecto-5'-nucleotidase (ecto-5'-NT, CD73), a (patho)biochemically important enzyme that hydrolyzes nucleoside-5'-monophosphates to the corresponding nucleosides. The enzymatic reaction was performed either before injection into the capillary (method A) or directly within the capillary (method B). In method A, separation of substrates and products was achieved within 8 min using an eCAP fused-silica capillary (20 cm effective length, 75 microM i.d., UV detection at 260 nm), 40 mM sodium borate buffer (pH 9.1), normal polarity, and a constant voltage of 15 kV. In method B, the sandwich technique was applied; substrate dissolved in reaction buffer (10mM Hepes [pH 7.4], 2mM MgCl2, and 1mM CaCl2) was hydrodynamically injected into a fused-silica capillary (30 cm, 75 microM i.d.), followed by enzyme (recombinant rat ecto-5'-NT) and subsequent injection of substrate solution. The reaction was initiated by the application of 1 kV voltage for 1 min. The voltage was turned off for 1 min and again turned on at a constant voltage of 15 kV to elute products (nucleosides) within 4 min using borate buffer (40 mM, pH 9.1). Thus, assays could be performed within 6 min, including enzymatic reaction, separation, and quantification of the formed nucleoside. The CE methods were used for measuring enzyme kinetics and for assaying inhibitors and substrates. In addition, the online assay was successfully applied to melanoma cell membrane preparations natively expressing the human ecto-5'-NT.     

Specific determination of intact cisplatin and monohydrated cisplatin in human plasma and culture medium ultrafiltrates using HPLC on-line with inductively coupled plasma mass spectrometry

We have developed a specific assay for cisplatin in human plasma ultrafiltrate (PUF) and cell culture medium ultrafiltrate (MUF) using HPLC on-line with inductively coupled plasma mass spectrometry (ICP-MS). Separation of cisplatin (6 min) and monohydrated cisplatin (12 min) was achieved using a muBondapak C(18) column (Waters) and a mobile phase (0.075 mM sodium dodecyl sulfate and 3% methanol, adjusted to pH 2.5 with triflic acid) pumped at a flow rate of 0.5 mL/min. The analytes were detected with little background interference by ICP-MS monitoring of platinum masses (m/z 194/195). Calibration curves were linear over three orders of magnitude (0.05-8 microM) and the limit of quantitation was 0.1 microM. Intra- and inter-assay accuracy (range 91.6-113%) and precision (range 1.00-12.3%) were acceptable for PUF and MUF. The method was applied to determining cisplatin during ex vivo incubation of the drug in whole human blood at 37 degrees C. In conclusion, a specific, sensitive and reliable HPLC-ICP-MS assay has been established for determining intact cisplatin in PUF and MUF.