High-performance liquid chromatographic determination of phenylephrine and its conjugates in human plasma using solid-phase extraction and electrochemical detection

An HPLC method for the determination of phenylephrine and its conjugates in human plasma was developed and validated. The method for quantitation involved extraction of diluted plasma (subject to hydrolysis with β-glucuronidase for 30 min with 500 units of enzyme per 0.1 ml of plasma at 37°C for the conjugates) on solid-phase weak cation-exchange cartridges followed by elution of the analyte and the internal standard (ethylnorphenylephrine) with 5% triethylamine in methanol. Analysis was carried out on a 15 cm ODS stationary phase using ion-pair reversed-phase chromatography. An electrochemical detector operated at + 1.15 V vs. Ag/AgCl was employed for detection. The standard curves were linear in the range 1.0–50.0 ng ml⁻¹ for phenylephrine and 25.0–500.0 ng ml⁻¹ for phenylephrine obtained from its conjugates. The limit of quantitation was 2.0 ng ml⁻¹ (RSD = 17%) and 25.0 ng ml⁻¹ (RSD = 18%), respectively. Acceptable accuracy and precision were obtained during intra- and inter-batch analyses for both the assays.     

Simultaneous determination of dextrorphan and guaifenesin in human plasma by liquid chromatography with fluorescence detection

A sensitive liquid chromatographic (LC) method was developed and validated for the simultaneous determination of dextrorphan and guaifenesin in human plasma using fluorescence detection. Dextrorphan and guaifenesin were extracted from plasma by a liquid-liquid extraction procedure using chloroform containing laudanosine as the internal standard. A cyano column (15 cm × 46 mm i.d., Spherisorb 5-CN) and a mobile phase containing acetonitrile-triethylamine-distilled water (10:1:89, v/v/v) (pH 6) were used. The concentration-response relationship for dextrorphan was found to be linear over a concentration range of 23–515 ng ml⁻¹ with a lower limit of detection of 20 ng ml⁻¹; the accuracy of the method would fall (95% confidence limit) within 9.53% and 11.07% of the true value for the inter-and intra-day, respectively; the inter- and intra-day precision, as measured by RSD, ranged from 1.88% to 30.07% (mean 2.28%) and from 4.69% to 7.51% (mean 5.67%) over the dynamic concentration range of the method (33–326 ng ml⁻¹). The concentration-response relationship for guaifenesin was found to be linear over a concentration range of 181–8136 ng ml⁻¹ with a lower detection limit of 30 ng ml⁻¹; the accuracy of the method would fall (95% confidence limit) within 9.78% and 8.04% of the true value for the inter- and intra-day, respectively; the inter- and intra-day precision, as measured by the RSD, ranged from 2.55 to 6.07% (mean 3.90%) and from 3.12 to 3.90% (mean 3.52%) over the dynamic concentration range of the method (435–6430 ng ml⁻¹). The relative percentage recovery of dextrorphan, guaifenesin, and laudanosine was found to be 96%, 94%, and 88%, respectively. Benchtop and storage stability of the plasma samples were found to be adequate. In addition, the frozen plasma samples were submitted to three filter freeze/thaw cycles without significant change in the stability of guaifenesin and dextrorphan.     

Simplified reversed-phase HPLC method with spectrophotometric detection for the assay of verapamil in rat plasma

A high-performance liquid chromatographic (HPLC) method was developed for the assay of verapamil in rat plasma. After deproteinization of the plasma sample with an acetonitrile-perchloric acid (8:2) mixture containing dextromethorphan, the internal standard, an aliquot of the supernatant was directly analyzed on a cyanopropylsilane column with methanol-acetonitrile-triethylamine acetate buffer (10:30:60) as the mobile phase and detection at 235 mm. At a flow rate of 1.5 ml min⁻¹, a complete analysis was completed in less than 6 min. The method was linear for verapamil concentrations in the range 0.5–10 μg ml⁻¹ (r=0.9999). Recoveries for the same drug concentrations from spiked rat plasma ranged from 85.6-93.0% (n=8). The mean RSD values for intraday and interday assay reproducibility (n=3) were, in both cases, less than 0.9%. The limit of detectability was about 0.1 μg ml⁻¹. The method was found useful to monitor the plasma levels of verapamil in rats that had received this drug by the nasal, oral and intravenous routes of administration.     

A capillary gas-liquid chromatographic method for the assay of the neuroleptic drug zotepine in human serum or plasma

A capillary gas-liquid chromatographic method suitable for the assay of the atypical neuroleptic drug zotepine in human serum or plasma was developed. A liquid-liquid extraction with three subsequent extraction steps was applied for sample preparation. The minimum detectable concentration was 1.0 ng ml⁻¹. The within-day relative standard deviation (RSD) (n = 6) was 5.3% at 5 ng ml⁻¹, 3.6% at 10 ng ml⁻¹ and 6.1% at 100 ng ml⁻¹. The day-to-day RSD (n = 6) was 9.3% at 10 ng ml⁻¹ and 5.1% at 100 ng ml⁻¹. Steady-state serum levels of four schizophrenic patients were measured.     

HPLC assays to simultaneously determine the angiotensin-AT1 antagonist losartan as well as its main and active metabolite EXP 3174 in biological material of humans and rats

Novel rapid and sensitive HPLC assays were developed to simultaneously determine losartan and its main active metabolite EXP 3174 in biological material of humans and rats following solid–phase or liquid–liquid extraction. The analytes were separated on a 3 μm particle-sized ULTREMEX™ CN column, which was preceeded by a 5 μm particle-sized guard column, using UV-detection at 245 nm. The assays provided high sensitivity with limits of quantification (LoQ) of 5 ng ml⁻¹ for both compounds in human and rat plasma and 10 ng ml⁻¹ in human and rat urine, respectively. In rat blood, bile and various tissues, limits of quantifications were achieved that ranged 10–15 ng per ml and per 100 mg tissue, respectively, for both analytes.     

Analysis and pharmacokinetics of olanzapine (LY170053) and two metabolites in rat plasma using reversed-phase HPLC with electrochemical detection

A sensitive HPLC assay for measurement of the antipsychotic drug, olanzapine, in plasma has been developed. The assay has a limit of quantitation of 1 ng ml⁻¹ in plasma and utilizes solid-phase extraction and electrochemical detection. The method provides a linear response for olanzapine over a concentration range of 1–100 ng ml⁻¹ with coefficients of determination greater than 0.9912. The inter-assay precision was 15.9% at the limit of detection and ranged from 7.33% to 8.47% over the range of 5–100 ng ml⁻¹. The intra-assay precision was in the range 0.97%–26.0%. The inter-assay accuracy ranged from 98.9 to 118% and the intra-assay accuracy ranged from 92.5% to 125% of the theoretical value. In addition, the assay was extended to measure the plasma levels of two metabolites of olanzapine, namely the N-desmethyl- and the 2-hydroxymethyl analogs. The utility of the assay was demonstrated following the administration of a single oral dose of ¹⁴C-olanzapine to rats where, at several time-points after dosing, the plasma was assayed for total radioactivity, levels of olanzapine, and the two metabolites. Olanzapine and two of its metabolites accounted for less than 50% of the total plasma radiocarbon; olanzapine accounting for approximately 39% at the C_max, N-desmethyl for 5% and 2-hydroxymethyl for 8% respectively. The plasma elimination half-times for olanzapine and the two metabolites were approximately the same, ranging from 3.3 to 4.4 h.     

The determination in human plasma of 1-hydroxy-2-naphthoic acid following administration of salmeterol xinafoate

The clinical development of salmeterol xinafoate, the 1-hydroxy-2-naphthoic acid salt of salmeterol, a potent long acting β₂ agonist bronchodilator, has required the development of a method for the determination of 1-hydroxy-2-naphthoic acid (HNA), in human plasma. A sensitive, accurate and precise method was, therefore, required to enable the pharmacokinetic profile to be established. HNA was determined in human plasma using a semi-automated procedure with solid-phase extraction using an automated analytical sample processor (AASP) and high-performance liquid chromatography (HPLC) with fluorescence detection. The method was sensitive to 10 ng ml⁻¹. The method is specific for HNA with respect to endogenous plasma components and has been shown to be robust, accurate and precise. Over four independent assay runs, the relative standard deviations (RSD) of the quality control samples (QC) were 1.6, 2.4 and 5.5% at 180, 100 and 40 ng ml⁻¹, respectively. A pharmacokinetic profile of HNA in man has been established from a single dose kinetic study in healthy volunteers following an oral dose of 500 μg salmeterol xinafoate, equivalent to 225 μg HNA. Maximum plasma concentrations attained at 1 h after dosing ranged between 35.3 and 66.8 ng ml⁻¹ and were within the calibration range of the assay.     

Trace quantitation of the novel cholinesterase inhibitor in human plasma by capillary gas chromatography/ion trap mass spectrometry

This paper demonstrates the utility of an ion trap mass spectrometer as a detector for trace quantitative determinations of pharmaceuticals in human plasma by capillary gas chromatography/mass spectrometry. A novel acetylcholinesterase inhibitor (CI-1002) was selected as an illustrative example for the technique. When coupled with a selective solid-phase extraction, this approach was capable of quantifying as little as 34 pg (0.50 ng ml⁻¹, RSD = 12.7%) of compound on the column, and the inter-run precision was typically 3–4% RSD over a 0.5–25 ng ml⁻¹ linear range. The advantages and requirements of the technique, in addition to the propects for improvements in the detection limit, are discussed.     

A rapid liquid chromatographic method for the determination of lamotrigine in plasma

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method for the determination of lamotrigine in plasma is described. The drug was extracted from 100 μl of plasma with chloroform:isopropanol (95:5% v/v) in the presence of 100 μl of phosphate buffer (10 mM). The extract was evaporated and the residue was reconstituted with mobile phase and injected onto the HPLC system. The drug and the internal standard (chloramphenicol) were eluted from a Symmetry C₁₈ stainless steel column at ambient temperature with a mobile phase consisting of 0.01 M potassium phosphate–acetonitrile–methanol (70:20:10% v/v/v), adjusted to pH 6.7, at a flow rate of 1.3 ml min⁻¹ and the detector was monitored at 214 nm. Quantitation was achieved by measurement of the peak-area ratio of the drug to the internal standard and the lower limit of detection for lamotrigine in plasma was 20 ng ml⁻¹. The intraday precision ranged from 3.34 to 6.12% coefficient of variation (CV) and the interday precision ranged from 2.15 to 8.34% CV. The absolute and relative recoveries of lamotrigine ranged from 86.93 to 90.71% and from 95.18 to 107.13%, respectively. The method was applied in studying the pharmacokinetics of lamotrigine administered orally to rabbits. This reliable micro-method would have application in pharmacokinetic studies of lamotrigine where only small sample sizes are available, e.g. paediatric patients.     

HPLC determination of ketoprofen enantiomers in human serum using a nonporous octadecylsilane 1.5 μm column with hydroxypropyl β-cyclodextrin as mobile phase additive

A sensitive and stereospecific high-performance liquid chromatography (HPLC) method for the quantitation of ketoprofen enantiomers in human serum was developed. The assay involves the use of an octadecylsilane solid-phase extraction for serum sample clean-up prior to HPLC analysis. Chromatographic resolution of the ketoprofen enantiomers was performed on a nonporous octyldecylsilane column with hydroxypropyl β-cyclodextrin as the mobile phase additive. The composition of the mobile phase was 98:2 v/v aqueous 0.1% trifluoroacetic acid (TFA), pH 4.00 (adjusted with triethylamine (TEA))/acetonitrile containing 10 mM hydroxypropyl β-cyclodextrin (β-CD) at a flow rate of 0.8 ml min⁻¹. Recoveries of R(−)-ketoprofen was 95.4±2.16% and for S(+)-ketoprofen 96.2±1.31%. Linear calibration curves were obtained in the range 0.025–15 μg ml⁻¹ range for each enantiomer in serum. The detection limit based on a S/N=3 ratio was 10 ng ml⁻¹ for each enantiomer in serum with ultraviolet detection at 220 nm. The limit of quantitation for each enantiomer was 25 ng ml⁻¹. Precision calculated as % relative standard deviation (%R.S.D.) and accuracy calculated as % error were in the range 0.2–5.2% and 0.3–2.2%, respectively, for the R enantiomer and 0.3–6.2 and 0.2–3.2%, respectively, for the S enantiomer.     

Comparison of two extraction methods for determination of propranolol and furosemide in human plasma by mixed-mode chromatography

An isocratic high performance liquid chromatographic method is described for the determination of the β-adrenergic blocking drug, propranolol, and the diuretic, furosemide, in human plasma. The two compounds and the internal standard were extracted from plasma using a two-step extraction technique. Propranolol and pindolol (internal standard) were first extracted from alkaline plasma into diethyl ether; this was followed by extraction of furosemide into acidified ether: hexane (65:35). The two extracts were then combined and evaporated under nitrogen, and the reconstituted residues were analysed on a C18/SCX reversed-phase/cation exchange column with a mobile phase of acetonitrile: 0.1 M sodium acetate pH 4 (33:67). The drugs and the internal standard were detected by UV absorption at 230 nm. The drugs were also extracted from plasma by a column-switching technique utilizing a ten-port valve. The drug compounds were retained on a C18 pre-column. A comparison of RSD for within-batch (intra-assay) and between-batch (inter-assay) runs for both methods was carried out, the liquid/liquid extraction method giving better recovery values. The calibration graphs were linear from 25–300 ng ml⁻¹ for furosemide and 50–400 ng ml⁻¹ for propranolol. Recovery values were > 90.0% by liquid/liquid extraction and > 76.0% by column switching.     

Application of solid-phase extraction in the method development for determination of SEPA,an acronym for soft enhancement of percutaneous absorption,in human,rat, and rabbit serum using GC–FID method

A new nonaqueous topical minoxidil formulation containing SEPA (2-n-nonyl-1,3-dioxolane) for enhancement of percutaneous absorption was under evaluation. SEPA does not have chromophore for either ultraviolet or fluorescence detection using liquid chromatography and has no functional groups for derivatization. Therefore, a direct gas-chromatographic method with flame-ionization detection (GC–FID) was developed. Owing to the limited detection response of the FID detection, it needs a selective and concentrated extract for GC–FID analysis to improve the assay sensitivity to meet the requirement for pharmacokinetic evaluation after topical application. In addition, SEPA is a very volatile compound. Any extraction procedures involving evaporation will result in a poor recovery. The application of solid-phase extraction (SPE) makes it possible to achieve a selective and a 10-fold concentrated extract with an absolute extraction recovery of approximately 90%, which greatly improved the assay sensitivity. This method involved the extraction of SEPA and the internal standard (2-n-heptyl-1,3-dioxolane) from serum (0.1–1 ml) with 100 μl of hexane-chloroform (1:1, v:v) using a 50 mg 1.0 ml⁻¹ phenyl SPE column (Varian, Harbor City, CA, USA), followed by direct GC–FID analysis on a fused-silica column chemically bonded with cross-linked methyl silicone gum phase (Hewlett Packard Ultra-1, 12 m×0.2 mm×0.33 μm, Avondale, PA, USA). The assay demonstrated a lower limit of quantitation of 2.5 ng ml⁻¹ and a linear range of 2.5 to 250 ng ml⁻¹ with intra- and inter-assay precision and accuracy of ≤10%.     

A sensitive assay for the simultaneous measurement of alfentanil and fentanyl in plasma

A reversed-phase high-performance liquid chromatography method for the simultaneous determination of plasma concentrations of the narcotic analgesics alfentanil and fentanyl using papaverine hydrochloride as the internal standard is presented. Chromatographic separations were achieved with an Econosphere CN, 5 μm, 25 cm × 4.6 mm i.d. column and the effluent was monitored at 195 nm. The assay was linear over the clinically relevant plasma range of 2–2000 ng ml⁻¹ for alfentanil and 2–100 ng ml⁻¹ for fentanyl and has the sensitivity and specificity necessary to determine plasma concentrations of these compounds. Inter- and intra-day precision (RSD) for both compounds did not exceed 10% in these ranges. The assay procedure was utilized for pharmacokinetic studies of plasma concentrations in subjects receiving alfentanil and fentanyl during and after cardiac surgery. This will allow better elucidation of pharmacokinetic variables in this populace.     

Simultaneous quantitation of buspirone and its major metabolite 1-(2-pyrimidinyl)piperazine in human plasma by high-performance liquid chromatography with coulometric detection

Buspirone is a member of the azapirone group of anxiolytic drugs and has one major metabolite, 1-(2-pyrimidinyl)piperazine (1-PP). The analyte, its metabolite and the internal standard were extracted from plasma utilizing solid-phase extraction columns. Chromatography was performed using isocratic reversed-phase high-performance liquid chromatography with coulometric end-point detection. The calibration graph was linear over the range 0–50 ng ml⁻¹ of plasma. The lower limits of quantitation for buspirone and 1-PP were 0.5 and 2 ng ml⁻¹, respectively, when 1 ml of plasma was extracted. The intra-assay relative standard deviations (RSD) over the range of the calibration graph varied from 4 to 12.5% for buspirone and 1-PP. The inter-assay RSD was 6.9% for 1-PP and 9.6% for buspirone. The recovery averaged 96% for buspirone and 66% for 1-PP. Plasma profiles of buspirone and 1-PP following oral dosing are presented.     

Clinical analysis of sampatrilat,a combined renal endopeptidase and angiotensin-converting enzyme inhibitor: I: assay in plasma of human volunteers by atmospheric-pressure ionisation mass-spectrometry following derivatisation with BF3–methanol

Sampatrilat is a dual inhibitor of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) under development for the treatment of hypertension and congestive heart failure. In order to support the early clinical development (with oral administration and an expected low bioavailability), a sensitive and selective assay was required. A method for plasma was developed and validated employing HPLC–APCI–MS–MS. The plasma samples were extracted on solid-phase extraction cartridges, derivatised with BF₃–methanol, diluted, extracted again and then subjected to HPLC–APCI–MS–MS. Derivatisation was necessary because the two carboxyl group in the molecule prevented efficient ionisation in the heated nebuliser source. The calibration range was from 0.5 to 20 ng ml⁻¹ and the lower limit of quantification was 0.5 ng ml⁻¹. Imprecision and inaccuracy were determined on three separate occasions at three concentrations (0.5, 5 and 20 ng ml⁻¹) and shown to be lower than 10% in every case.     

Simultaneous quantitation of pioglitazone and its metabolites in human serum by liquid chromatography and solid phase extraction

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of pioglitazone (U-72107) and its potential metabolites (M-1 to M-6) in human serum was developed. The method involved a solid phase extraction (SPE) of pioglitazone, its metabolites, and the internal standard (U-92573) from serum using C₁₈ SPE columns with an elution solvent of 0.5 ml of acetonitrile-water (35:65, v/v). Separation of the eight analytes was achieved within 20 min using a reversed-phase Zorbax RX-C₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size) with a mobile phase of acetonitrile-water (40:60, v/v) containing 3 ml acetic acid per liter mobile phase (apparent pH 5.5). An ultraviolet detector operated at 269 nm was used with a linear response observed from 0.02 to 2 μmg ml⁻¹ for these analytes except for M-4 which was best fitted with a polynomial regression. Limit of quantitation was found to be 0.02 μg ml⁻¹ for pioglitazone, M-3, M-5, and M-6; 0.04 μg ml⁻¹ for M-2 and M-4; and 0.5 μg ml⁻¹ for M-1 when using a 0.5 ml serum sample for extraction. Obtained from the method validation, intra- and inter-assay precision was ⩽9% and accuracy ranged from −8.2 to 13.4% for all analytes. The applicability of this method has been demonstrated by successfully analyzing clinical serum samples. The strategies in the HPLC characterization and in the SPE procedure development for this method are discussed as well.     

Determination of PNU 153429, a new polysulphonated derivative of distamycin A,in rat plasma by reversed-phase ion-pair high-performance liquid chromatography with ultraviolet detection

A rapid and selective ion-pair high-performance liquid chromatographic (HPLC) method for the determination of 2,2′-(carbonylbis(imino-N-methyl-4,2-pyrrolecarbonylimino(N-methyl-4,2-pyrrole)carbonylimino))-bis(1,5-naphtalenedisulphonic acid), tetrasodium salt (PNU 153429, I) in rat plasma has been developed. I is a new drug currently under investigation for the treatment of rheumatoid arthritis. Aliquots of 100 μl of plasma spiked with 10 μl of internal standard solution (PNU 145156E, I.S.) were added to 100 μl of acetonitrile and vortex mixed. After centrifugation, diluted aliquots of the supernatant were transferred to autosampler vials and analyzed by reversed-phase ion-pair liquid chromatography under isocratic conditions. The retention times of I.S. and I were ≈8 and 12 min, respectively. Quantitation was achieved by ultraviolet detection at 323 nm. The assay had a limit of quantitation of 0.1 μg ml⁻¹ when 100 μl of plasma were analyzed. The linearity, precision and accuracy of the method were evaluated. No interference from blank rat, mouse, dog, monkey and human plasma was observed. The suitability of the method for in vivo samples was checked by analysis of plasma samples drawn from three cannulated male rats that had received a single 100 mg kg⁻¹ i.v. dose of the test compound.     

Specific radioimmunoassays for the measurement of stavudine in human plasma and urine

Sensitive and specific radioimmunoassays (RIAs) have been developed and validated for the determination of stavudine, a nucleoside analog possessing anti-human immunodeficiency virus (HIV) activity, in human plasma and urine. The hemisuccinate of stavudine was conjugated with histamine and radioiodinated to yield the radiotracer. Antisera were produced by injecting the immunogen, stavudine-hemisuccinate-bovine thyroglobulin, into rabbits. The antisera exhibited high specificity for stavudine as the structurally related analogs and other anti-HIV agents did not interfere in the assays. The methods could reliably quantitate stavudine in plasma from 2.5–100 ng ml⁻¹ and in urine from 5.0–1000 ng ml⁻¹ (after 2.5-fold dilution) with good accuracy and precision. The lower limits of quantitation were 2.5 ng ml⁻¹ in human plasma and 5.0 ng ml⁻¹ in urine (after 2.5-fold dilution). The RIA methods were applied to the analysis of stavudine in plasma and urine obtained from HIV-infected patients receiving the drug in clinical trials.     

Sensitive determination of erythromycin in human plasma by LC-MS/MS

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of erythromycin in human plasma (EDTA as anticoagulant) was developed and validated. The concentration ranges were 0.5–50 and 50–5000 ng ml⁻¹. The procedure involved alkalization of 0.5 ml of plasma, one step liquid-liquid extraction, dryness of the extract and reconstitution in 80:20 water:acetonitrile. An Inertsil ODS-2 5 μm, 3.0×50 mm column (Metachem) with a C₈ guard column and isocratic mobile phase were used for liquid chromatography. The mobile phase consisted of 1:1 acetonitrile:water with 2 mM NH₄OAc and 0.1% HOAc. A flow rate of 0.7 ml min⁻¹ was used. The analysis time on LC-MS/MS for one sample was ≈2 min. A Turbo-Ionspray source was interfaced between the HPLC and triple quadrupole mass spectrometer (Sciex API III Plus). MS/MS analysis used Multi-Reaction Monitoring (MRM) mode. The lowest limit of quantitation (LOQ) was 0.5 ng ml⁻¹ with all Quality Control (QC) sample recoveries varying between 88 and 105%. Nine intraday and interday calibration curves were generated yielding correlation coefficients ranging from 0.995 to 1.000. Average recovery for erythromycin at 1 ng ml⁻¹ was 105% (±4.5%). Average recovery for the internal standard was 83–103%. Short-term and long-term stability in the freezer (−20°C), bench stability, and stability after 3 freeze/thaw cycles at −20 and −80°C were conducted. The samples were found to be stable under all conditions. The method developed and validated proved useful for clinical pharmacokinetic study sample analysis with high throughput due to its high sensitivity and very short analysis time.     

The development of a sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A receptor antagonist, in human plasma

A sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A (CCK-A) receptor antagonist, was developed to study the pharmacokinetics of the drug at low-dose administration using a specific monoclonal antibody. The high performance liquid chromatography (HPLC) method had been used for studying toxicokinetics, but its determination limit (2.5 ng ml⁻¹) was too high for use in clinical studies. Subsequently we developed an enzyme immunoassay (EIA) using rabbit anti-FK480 serum (polyclonal antibody). It had higher sensitivity (0.1 ng ml⁻¹) when 0.5 ml of plasma was used but its specificity was low because of the cross-reactivity of the metabolites of FK480. Therefore we produced several monoclonal antibodies for FK480 by cell fusion, and selected the antibody which was least cross-reactive for the isolated metabolites of FK480. Finally we developed a sensitive and specific EIA using this monoclonal antibody. The lower limit of quantification of this method was 0.2 ng ml⁻¹ when 0.2 ml of human plasma was used. The coefficient of variation over the calibration range (0.2–10 ng ml⁻¹) was less than 15%. We used this method for clinical studies, and it showed a good correlation to the HPLC method when plasma concentration was 2.5 ng ml⁻¹ or more.