Comparison of the fluorescence polarization immunoassay and the microbiological assay methods for the determination of gentamicin concentration in human serum

The performance of the fluorescence polarization immunoassay (FPIA) was compared with that of a microbiological assay for the measurement of serum gentamicin concentrations. Within-run precision from duplicate assays of two concentrations (4 and 8 micrograms/ml) using FPIA and the microbiological assay yielded coefficients of variation (r) of 2.62%, 1.76% (n = 12) and 8.06%, 6.87% (n = 12), respectively. Day-to-day precision was estimated by repetitive analysis of 4 and 8 micrograms/ml control samples over a 3-week period. Coefficients of variation (r) were 2.57%, 3.09% (n = 8) and 10.71%, 14.20% (n = 8) for FPIA and the microbiological assay, respectively. Linear regression analysis performed on data from parallel determinations on 143 patient samples by the two methods showed correlations in the order of 0.74. The FPIA offers a rapid, efficient, and accurate system for therapeutic monitoring of gentamicin serum levels.     

Evaluation of a fluorescence polarization immunoassay procedure for quantitation of methotrexate

A fluorescence polarization immunoassay (FPIA) procedure for measuring methotrexate was evaluated. The dynamic range of the assay is from 0.05 to 810 microM, and the calibration curve can be stored for at least 2 weeks. The FPIA procedure is automated and rapid; one result can be obtained in 18 min and five results in 25 min. There was no interference from hemoglobin (800 mg/dl), triglycerides (500 mg/dl), bilirubin (20 mg/dl), and protein (12.1 g/dl). Cross-reactivity with 7-hydroxy methotrexate and 2,4-diamino-N-methylpteroic acid was 0.6 and 44%, respectively. The coefficient of variation for the within-run and between-run precision was less than 5.0%. For the comparison studies, the samples were divided into four groups. The methotrexate concentrations in group 1 were 0.05-2.1 microM; in group 2, 2.2-9.3 microM; in group 3, 10-80 microM; and in group 4, greater than 80 microM. Linear regression analysis of the results obtained with the FPIA procedure and the enzyme multiplied immunoassay gave a correlation coefficient of at least 0.95 for all groups.     

Teicoplanin measurement in patients with renal failure: comparison of fluorescence polarization immunoassay, microbiological assay, and high-performance liquid chromatographic assay.

Characterization of antibiotic pharmacokinetics in patients with renal insufficiency may be complicated by interfering substances within the assay. We compared three different assays for teicoplanin in serum and dialysate of 10 hemodialysis and six continuous ambulatory peritoneal dialysis (CAPD) patients. The microbiological assay (micro) had a within-run and between-run coefficient of variation (% CV) of less than 7.5% for concentrations ranging from 0.2 to 96 micrograms/ml. The high-performance liquid chromatographic assay (HPLC) within- and between-run %CV was less than 8% for concentrations ranging from 1 to 80 micrograms/ml. The fluorescence polarization immunoassay (FPIA) within- and between-run %CV was less than 7% for concentrations ranging from 5 to 100 micrograms/ml. In serum of hemodialysis patients FPIA results were slightly higher than HPLC results: FPIA = 1.11 HPLC + 2.37 (r = 0.975, n = 202), and FPIA concentrations in serum were also slightly higher than those measured by micro (FPIA = 1.21 micro - 1.57, r = 0.972, n = 161). The HPLC and micro serum results were also comparable in hemodialysis patients: micro = 0.92 HPLC + 2.89, r = 0.953, n = 160. However, in CAPD patients micro results were lower than HPLC results in serum (micro = 0.82 HPLC + 0.49, r = 0.981, n = 262). In peritoneal dialysate, HPLC values were approximately 60% of the micro values. Thus, FPIA may be the optimal technique for therapeutic monitoring of teicoplanin in the clinical setting due to its simplicity, specificity, and good correlation to HPLC and micro.     

Comparison of particle-enhanced turbidimetric inhibition immunoassay and fluorescence polarization immunoassay using monoclonal antibodies for theophylline

The DuPont theophylline assay reagent kit, a particle-enhanced turbidimetric inhibition immunoassay (PETINIA) method adapted for use on a centrifugal fast analyzer, was evaluated. It was compared with fluorescence polarization immunoassay (FPIA). Day-to-day precision was 4.7% at 6.8 micrograms/ml, and 3.3% at 26.6 micrograms/ml. The assay is linear to a concentration of 40 micrograms/ml. Good correlation was found between the two methods (PETINIA/FPIA: y = 1.04x + 0.15, r = 0.988, Syx = 1.19) in the evaluation of 176 patients receiving theophylline. This method offers a precise and accurate alternative to FPIA.     

Evaluation of the powder-formulated enzyme multiplied immunoassay technique quantitative single test for gentamicin

The usefulness of the enzyme multiplied immunoassay quantitative single test (EMIT QST) gentamicin assay was assessed for gentamicin analysis in patient sera. The EMIT QST reagents are in powder form in a single, premeasured vial and are run on a thermoregulated sample processor that controls mixing and timing steps. The results of the clinical evaluation showed that the standard curve was stable throughout a 26-day study period. Within-run precision on 20 replicates at 4.0 micrograms/ml yielded a coefficient of variation (CV) of 5.6%; between-run precision on 66 analyses at 6.0 micrograms/ml over a 152-day period yielded a CV of 4.0%. Mean recovery through the range of the standard curve with 10 spiked patient samples was 102%. Comparative analysis with radioimmunoassay of 95 patient samples showed a correlation of 0.97, with y = 0.93x - 0.03. It was concluded that the EMIT QST gentamicin assay is an appropriate, rapid methodology for patient gentamicin analysis.     

Rapid sample preparation and high performance liquid chromatographic determination of total and unbound serum disopyramide

A rapid sample preparation procedure, which requires no solvent extraction or concentration, for the high performance liquid chromatographic (HPLC) determination of disopyramide is described. The chromatography is performed on a C-18 radial compression mu-Bondapak column and detection by absorbance at 254 nm with a run time of 12 min. The mobile phase is 10 mM sodium acetate (pH 4.5)/acetonitrile (3:1 vol/vol). For the total drug assay, 50 microliter 30% (wt/vol) trichloroacetic acid is added to 500 microliter serum, which causes the precipitation of protein. Following centrifugation, 100 microliter of supernatant is mixed with 25 microliters of internal standard (25 micrograms/ml, ethyl p-aminobenzoate), and 50 microliters of this mixture is injected into the HPLC. Unbound disopyramide is separated from protein-bound drug by filtration with an Amicon Centrifree filter, which removes 99.6% of protein and does not retain disopyramide. To 100 microliters of this filtrate is added 25 microliters of internal standard, and 50 microliters is injected into the HPLC. The assay is linear to at least 20 micrograms/ml. The total drug assay shows an average recovery of 93.0% with an average coefficient of variation (CV) of 3.4%. The unbound drug assay shows an average CV of 4.1%. The percentage of free drug in a sample containing 4.85 g/dl protein varies from 68.0 to 83.5% at concentrations of 2.5-10 micrograms/ml, which illustrates the concentration-dependent nature of the protein binding, and the need to measure the unbound fraction of drug. Of 31 drugs tested for interference, none was found to interfere.     

Analytic performance evaluation of a new turbidimetric immunoassay for carbamazepine on the ADVIA 1650 analyzer: effect of carbamazepine 10,11-epoxide

Carbamazepine, an anticonvulsant, requires therapeutic drug monitoring. Recently Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of carbamazepine on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained with this new assay in sera of 54 patients receiving carbamazepine with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA) and a chemiluminescent immunoassay (CLIA). The new turbidimetric immunoassay for carbamazepine showed excellent precision. The low control showed a total CV of 4.9% (mean 2.86, SD 0.14 microg/mL), the medium control demonstrated a total CV of 3.5% (mean 7.79, SD 0.27 microg/mL), and the high control showed a total CV of 4.8% (mean 16.15, SD 0.78 microg/mL). The assay was linear up to a carbamazepine concentration of 20 microg/mL. The assay showed excellent dilution recovery and recovery of samples supplemented with carbamazepine (mean recovery 102.2%). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y = 0.96 x - 0.46, r = 0.99, n = 54). We also observed excellent correlation between the values obtained by the CLIA (x-axis) and the turbidimetric (y-axis) assay (y = 1.10 x -0.32, r = 0.99, n = 54). However, the slope of 1.10 was higher than the slope of 0.96 observed with the regression equation obtained by using values obtained by the FPIA and the turbidimetric assay. The positive bias obtained with the new turbidimetric assay compared with the CLIA assay resulted from lower cross reactivity of carbamazepine 10,11-epoxide, the active metabolite of carbamazepine, with CLIA. On the other hand, the cross reactivity of the metabolite is similar between the new turbidimetric assay and the FPIA assay. We conclude that the new turbidimetric assay can be used for routine monitoring of carbamazepine in clinical laboratories.     

Evaluation of a new phencyclidine enzyme immunoassay for the detection of phencyclidine in urine with confirmation by high-performance liquid chromatography-tandem mass spectrometry

We present an evaluation of a new phencyclidine (PCP) enzyme immunoassay (PCPI) for detection in urine. The PCPI was evaluated by testing 523 urine specimens. Controls containing 0 ng/mL of PCP and -25% (negative control) and +25% (positive control) of the 25 ng/mL cutoff calibrator were analyzed with each batch. All urines were analyzed by high-performance liquid chromatography- tandem mass spectrometry (HPLC-MS-MS) for PCP. Of the 523 specimens tested, 218 yielded positive results by the PCP assay. HPLC-MS-MS confirmed the presence of at least 25 ng/mL in 214 specimens, indicating four false-positive results containing 11, 13, 16, and 20 ng/mL PCP. Three specimens yielded a negative result; however, PCP concentrations were within 20% of the 25 ng/mL cutoff value. The overall agreement of PCPI and HPLC-MS-MS results was 98.7%. The sensitivity of the PCPI was 0.982 and the specificity 0.987. Testing at 100 ng/mL of other drugs or their metabolites demonstrated no cross-reactivity. The within-run precision was CV = 2% (n = 12); the between-run precision was CV = < 6% (n = 4). The assay was found linear from -50% to 150% (12.5-37.5 ng/mL) of the cutoff concentration. The Lin-Zhi PCPI provides a precise, reliable method for the routine detection of phencyclidine in urine specimens.     

Measurement of cyclosporine in plasma of cardiac allograft recipients by fluorescence polarization immunoassay

The Abbott TDx fluorescence polarization immunoassay (FPIA) procedure for measuring cyclosporine A (CsA) was evaluated and compared with the Sandoz polyclonal radioimmunoassay (CsA RIA kit) method. This drug assay was evaluated for precision, calibration, stability, and accuracy. Within-run precision studies utilizing 25 replicate analyses of the three control preparations (containing CsA in the 60-800 ng/ml range) resulted in coefficients of variation (CV) ranging from 1.0 to 9.1%. The CVs of between-run precision determined by assaying the same control drug levels for five consecutive working days ranged from 3.9 to 4.6%. Calibration curve stability was assessed by examining the drift in control values over a 2-week period. Maximum plasma ranged from 82.6 to 108.2%. Four hundred plasma samples were obtained from 30 heart-transplant patients during the first 6 months of CsA therapy and each sample was analyzed simultaneously by TDx and RIA. Linear regression analysis of the results obtained for each patient (x = RIA, y = FPIA) revealed the following mean values: r = 0.87, (CV = 13.7%), slope = 1.47 (CV = 39.2%). Moreover, the concentration of CsA was determined in 35 patient samples both by TDx and high-performance liquid chromatography (HPLC). FPIA results up to 12 times higher than HPLC results have been noted.     

A rate nephelometric phenobarbital and phenytoin assay: analytical and correlation studies of a new single-point calibration immunoprecipitation inhibition method

A new single-point calibration immunoprecipitation inhibition rate nephelometric assay for phenobarbital (PB) and phenytoin (PT) has been evaluated on the Beckman ICS-II analyzer. Within-run precision (n = 20) coefficients of variation (CV) were less than 4.0% for PB (10-44 micrograms/ml) and less than 5.7% for PT (12-34 micrograms/ml). Between-run precision CVs of pooled sera (n = 20) assay were less than 4.2% for PB (7.5-33.8 micrograms/ml) and less than 8.7% for PT (4.5-17.8 micrograms/ml). Linearity and recoveries were good over a broad range of drug levels for both assays. Excellent correlation was found with existing PB, ICS vs. EMIT on the IL Multistat III MCA (y = 1.26 + 1.00x; r = 0.994), ICS vs. Dupont aca (y = 0.832 + 1.01x; r = 0.997), and ICS vs. GLC (y = 0.08 + 0.99x; r = 0.998) methods, and PT, ICS vs. MCA (y = -1.76 + 1.04x; r = 0.985), ICS vs. aca (y = -2.02 + 1.054x; r = 0.987), and ICS vs. GLC (y = -0.26 + 0.990x; r = 0.999) methods. These assays were found to provide accurate drug analyses, good stat or batching capability, and good correlation with existing methods.     

Determination of isepamicin sulfate and related compounds by high performance liquid chromatography using evaporative light scattering detection

A simple reversed phase high performance liquid chromatographic method was developed for the analysis of isepamicin sulfate. The use of evaporative light scattering detection eliminates the need for sample derivatization. Separation of the isepamicin aminoglycoside from structurally similar related compounds was achieved using two Waters X-Terra RP18 columns connected in tandem at 10 degrees C. The assay of isepamicin sulfate and the estimation of its impurities was accomplished using external standard calibration curves at two sample concentrations: 1.6 mg ml(-1) for the analysis of isepamicin sulfate and 8.0 mg ml(-1) for the estimation of lower level impurities. The limit of detection was 0.1%. The specificity, assay linearity, low level assay linearity and assay repeatability were also investigated.     

Ex vivo protein binding of clindamycin in sera with normal and elevated alpha 1-acid glycoprotein concentrations.

Clindamycin is a lincosamide antibiotic that binds primarily to alpha 1-acid glycoprotein (AAG), an acute-phase serum protein. Many studies have shown that AAG concentrations increase in response to stress, including infection, myocardial infarction, and trauma. The objectives of this study were to determine the serum protein binding of various clindamycin concentrations in sera with normal and elevated AAG concentrations. Serum was obtained from 4 healthy volunteers and 12 patients with pathophysiologic conditions known to elevate serum AAG concentrations. Timing for collection was determined from the literature, corresponding with the expected peak concentration for each disease state. Samples were assayed for AAG by radial immunodiffusion and were spiked with clindamycin to achieve total concentrations of 10 micrograms/ml (n = 18), 4 micrograms/ml (n = 10), and 2 micrograms/ml (n = 7). Protein binding was determined by ultrafiltration and subsequent high-performance liquid or gas chromatography. Protein binding was dependent on the serum concentrations of both AAG and clindamycin. When AAG concentrations increased from 101-150 mg/dl to 201 mg/dl or greater, mean protein binding increased from 81.2% to 92.4% (p = 0.1265) and from 61.3% to 88.6% (p less than 0.05) at clindamycin concentrations of 2 and 4 micrograms/ml, respectively. With AAG concentrations between 101 and 150 mg/dl, mean protein binding increased from 62.4% at 10 micrograms/ml to 81.2% at 2 micrograms/ml (p = 0.1514). Since AAG concentrations may increase in certain patients, the concentration of free (pharmacologically active) drug may fall below the minimum inhibitory concentration for several pathogens earlier in a dosing interval.     

A dry-strip immunometric assay for digoxin on the Ames Seralyzer

We evaluated the Ames Seralyzer III with a new reagent and drystrip test for assay of digoxin. Assay precision was acceptable in the therapeutic range. Within-run imprecision (coefficient of variation, n = 20) was 7% at 0.9 ng/ml (1.2 nM) and 3.5% at 1.9 ng/ml (2.4 nM); run-to-run imprecision was 7.6% at 0.8 ng/ml (1.0 nM) and 5.7% at 2.1 ng/ml (2.7 nM). The method is very reproducible and is linear between 0.5 and 4.3 ng/ml (0.6-5.5 nM). The assay performed well with patient samples, with Abbott TDx used as the reference procedure. Bilirubin up to 16 mg/dl (273 microM) and hemoglobin up to 11 g/l do not cause interference. Digoxin-like immunoreactive factors cause minimal interference. Some digoxin metabolites such as monodigitoxoside, bis-digitoxoside, and digoxigenin cross-react with the digoxin antibody. Patients on spironolactone have falsely increased digoxin values. The new digoxin assay is easy to perform and uses 30 microliters serum; the result can be reported in 15-20 min.     

Performance of a fluorescence polarization immunoassay system evaluated by therapeutic monitoring of four drugs.

Fluorescence polarization immunoassays (FPIA) for amikacin, gentamicin, quinidine, and theophylline (supplied by Roche Diagnostic Systems, made using a Cobas Fara centrifugal analyzer) were evaluated and compared with widely used monitoring analysis methods. For each drug, the between-assay imprecision was ascertained by calibration on the day of assay and by a stored calibration curve made at the beginning of the study. The precision of the amikacin and theophylline assays was acceptable [total coefficient of variation (CV) less than 7.5%] at all concentrations tested for each calibration mode. Imprecision of quinidine and gentamicin assays was significant at low concentrations (1.9 mg/L): total CV = 9.0% for quinidine assessed with stored calibration curve and total CV greater than 8.5% for gentamicin measured with the two calibration modes. The calibration curves for all four assays had a good stability (greater than 30 days). Linear regression analysis demonstrated close agreement between the FPIA (y) and the following comparative techniques (x): Abbott TDx assay for amikacin and gentamicin (r = 0.988, r = 0.974, respectively); Stratus fluorometric enzyme immunoassay for quinidine (r = 0.979); and EMIT Syva assay for theophylline (r = 0.993). It is concluded that fluorescence polarization immunoassay is a rapid and reliable method for the therapeutic monitoring of the four drugs tested. Moreover, the use of reagents on an instrument that can be implemented for a wide range of chemistries has significant advantages and cost benefits over dedicated instruments.     

Evaluation of the Ames Seralyzer for therapeutic drug monitoring of theophylline

Dry reagent technology and reflectance photometry are combined in the Ames Seralyzer to offer a solid-phase plastic strip assay methodology. Light reflected from serum placed on the antibody-impregnated strip is quantitated with a two-point standard curve to display a theophylline result in 90 seconds. The accuracy and precision of the Seralyzer for serum theophylline are compared to the enzyme-multiplied immunoassay technique (Emit) which is commonly used to determine drug concentrations. Liquid calibrators (5 and 25 micrograms/ml) and liquid-spiked sera (10, 15, and 20 micrograms/ml) were measured six times daily simultaneously by both methods for twenty days. Eighty patient samples (theophylline range: 0-33 micrograms/ml) were measured twice each during the twenty days. Acceptable assay limits were established and maintained by measuring 15 micrograms/ml spiked sera prior to and during the evaluation period. All within-run and between-run means for the Seralyzer and Emit were within +/- 1 microgram/ml of the spiked value. All ranges of within-run and between-run means were within +/- 2 micrograms/ml of the spiked value. Mean between-run coefficients of variation for the Seralyzer at the 5, 10, 15, 20, and 25 micrograms/ml values were 11.5, 6.7, 4.9, 4.6, and 4.9 percent, respectively. Linear regression on the 80 patient samples gave a slope of 1.02, intercept of -0.28, and correlation of 0.984. The Seralyzer's accuracy and precision compare favorably with Emit for theophylline determinations greater than 7.5 micrograms/ml.     

Evaluation of fluorescence polarization immunoassay for determination of cyclosporin in plasma

The fluorescence polarization immunoassay (FPIA) method for determination of cyclosporin in plasma was evaluated and compared with the high-performance liquid chromatography (HPLC) and the radioimmunoassay (RIA) methods. The coefficients of variation for the within-run and between-run precision were less than 5 and less than 8%, respectively, for samples ranging in concentration from 50 to 600 ng/ml. Recoveries were determined by adding cyclosporin at concentrations from 25 to 1,000 ng/ml to patient plasma; they were, on average, 98.5%. The calibration curve was stable throughout a 10-week study period. There was no clinically significant interference due to hemolysis, icterus, lipemia, or other commonly used drugs. There was considerable variation of the ratio of the FPIA result to the HPLC result, whereas there was a good correlation between the FPIA and the RIA results (r = 0.975, n = 25, y = 1.2x - 36.4), when evaluated using specimens from renal transplant patients receiving cyclosporin orally. It was concluded that the FPIA is an appropriate, rapid method for patient cyclosporin analysis in plasma and serves as a practical alternative to the RIA.     

Analytic performance evaluation of a new turbidimetric immunoassay for phenytoin on the ADVIA 1650 analyzer: effect of phenytoin metabolite and analogue

Phenytoin is an anticonvulsant that requires therapeutic drug monitoring. Recently, Bayer HealthCare, Diagnostics Division released a turbidimetric immunoassay of phenytoin on the ADVIA 1650 analyzer. We evaluated the analytic performance of this assay by comparing values obtained in 52 patients receiving Phenytoin using this new assay with the values obtained by using a widely used fluorescence polarization immunoassay (FPIA). The new turbidimetric immunoassay for phenytoin showed the following imprecision with the low, medium, and high controls: total CV of 5.2% (mean 4.81 microg/mL), 3.7% (mean 16.24 microg/mL), and 4.1% (mean 22.65 microg/mL), respectively. The detection limit of the assay was 0.79 microg/mL, and the assay was linear up to a phenytoin concentration of 46.1 microg/mL. The assay showed excellent dilution recovery and recovery of spiked samples (mean recovery 101.4% and 94.4%, respectively). We observed an excellent correlation between the values obtained by the FPIA (x-axis) assay and the new turbidimetric (y-axis) assay (y=1.06 x-0.61, r=0.98, n=52). We also determined the cross-reactivity of 5-(p-hydroxyphenyl)-5-phenylhydantoin (HPPH), a major metabolite of phenytoin, and of oxaprozine, an analogue with a similar chemical structure to phenytoin, in both phenytoin assays. Both assays showed almost no cross-reactivity to oxaprozine and only small (5%-8%) cross-reactivity to HPPH. We also found that the turbidimetric assay was free from interference at least up to 1200 mg/dL of hemolysis, 30 mg/dL of free bilirubin, 34.5 mg/dL of conjugated bilirubin, and 750 mg/dL of triglyceride (Intralipid). When a drug-free serum was followed by a serum sample containing 38.5 microg/mL of phenytoin, no sample probe carryover effect was observed. We conclude that the new turbidimetric assay can be used for routine monitoring of phenytoin in clinical laboratories.     

A study of the absorption and excretion of fosfomycin sodium in children

Fosfomycin sodium (FOM-Na, Forocyle-S) was administered at 25 mg/kg or 50 mg/kg to 15 children between the ages of 3 and 15 through intravenous injection or through 1 hour intravenous drip infusion, and concentrations in blood serum and excretion through urine were examined and a pharmacokinetic analysis was carried out using the one-compartment model. 1. Average concentrations in the blood serum after injections with 25 mg/kg and 50 mg/kg were 55.3 +/- 6.3 micrograms/ml and 118.8 +/- 31.1 micrograms/kg 30 minutes after injection, respectively, and their half-lives were 1.04 +/- 0.15 hours and 0.98 +/- 0.17 hours, respectively. Six hours after injection, the levels were 2.7 +/- 1.6 micrograms/kg and 6.2 +/- 5.5 micrograms/kg, respectively. With 1 hour intravenous drip infusion of 25 mg/kg and 50 mg/kg, average concentrations the blood serum were 34.2 +/- 14.9 micrograms/ml and 89.7 +/- 6.7 micrograms/ml, respectively, and their half-lives were 0.87 +/- 0.24 hour and 0.69 +/- 0.10 hour, respectively. Six hours after the administration, the levels were 2.7 +/- 1.8 micrograms/ml and 6.7 +/- 0.8 micrograms/ml. There was a clear dose response in the concentration levels in the blood in those given the drug at 25 mg/kg and 50 mg/kg in either method of administration. 2. Average levels in urine after injection of 25 mg/kg and 50 mg/kg were 5,778 +/- 2,257 micrograms/ml and 6,268 +/- 3,329 micrograms/ml 0-2 hours after administration, respectively, and average levels at 4-6 hours were 701 +/- 765 micrograms/ml and 1,588 +/- 1,324 micrograms/ml, respectively. Average excretion, rates into the urine were 72.8 +/- 11.0 and 73.9 +/- 11.1%, respectively. In case of 1 hour drips infusion of 25 mg/kg and 50 mg/kg, average concentrations in the urine 0-2 hours after administration were 3,570 +/- 1,540 micrograms/ml and 11,800 micrograms/ml, respectively, and averages for 4-6 hours were 211 +/- 124 micrograms/ml and 1,300 micrograms/ml. Average rates of excretion into the urine for the first group was 57.9 +/- 16.3% and the second group was 78.4%. Clear dose response was observed in changes of drug concentration levels in the urine with 25 mg/kg and 50 mg/kg doses through either administration method, and in terms of excretion into the urine, no noticeable differences were observed between the different amounts administered or different administration methods.(ABSTRACT TRUNCATED AT 400 WORDS)     

A high-performance liquid chromatography-mass spectrometry method using a novel atmospheric pressure chemical ionization approach for the rapid simultaneous measurement of tacrolimus and cyclosporin in whole blood

Concentration monitoring and dose individualization is required to optimize either tacrolimus or cyclosporin therapy. In this study, the validation of a simple, rapid high-performance liquid chromatography-tandem mass spectrometry method for the simultaneous measurement of tacrolimus and cyclosporin in whole blood is reported. Blood samples (100 microL) were prepared by protein precipitation with zinc sulphate followed by acetonitrile (containing the internal standards ascomycin and cyclosporin D). The chromatographic run time was 1.5 minutes per sample. Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in negative ionization mode (tacrolimus: m/z 802.5 --> 560.6, cyclosporin: m/z 1200.8 --> 1088.4). The assay had an analytical range of 1.0 to 30 mug/L (r > 0.998, n = 6) for tacrolimus and 25 to 2000 microg/L (r > 0.999, n = 6) for cyclosporin. Tacrolimus inter- and intraday inaccuracy and precision [coefficient of variation (CV)] using quality control samples (2.5, 12.5, 25 microg/L) was less than +/-10.0% and CV less than 5.0%, respectively (n = 5). Similarly, cyclosporin inter- and intraday inaccuracy and precision using quality control samples (70, 400, 1500 microg/L) was less than +/-2.0% and CV less than 5.0%, respectively (n = 5). The lower limit of quantification for tacrolimus was 1.0 mug/L and cyclosporin 25 microg/L. The assay had an absolute mean recovery of 86.7% for tacrolimus and 89.0% for cyclosporin (n = 15). Intersubject variability, as a measure of potential matrix effects on results, was less than 6.0% CV for both analytes (n = 15). Extracted samples were stable for at least 20 hours. Results obtained from external proficiency testing samples measured by this method compared with the mean of all liquid chromatography-tandem mass spectrometry methods used by scheme participants revealed a strong correlation and good agreement for tacrolimus (r = 0.993, mean bias = -10.3%, n = 19) and cyclosporin (r = 0.996, mean bias = 3.0%, n = 20). In conclusion, this is the first reported high-throughput method that uses negative atmospheric pressure chemical ionization for the simultaneous measurement of tacrolimus and cyclosporin in whole blood.     

Liquid chromatographic determination of piroxicam in serum

A sensitive method for monitoring serum piroxicam (Feldene) is described. The assay requires 1.0 ml of specimen and involves chloroform extraction from an acidified mixture followed by concentration and injection into a liquid chromatograph. Column effluent is monitored at 330 nm. Retention times of piroxicam and the internal standard (naproxen) are 6.6 and 11.0 min, respectively. The lower limit of detection in serum is 0.5 mg/L. Within-day precision (coefficient of variation, CV) of piroxicam in serum (5-13 mg/L range) varied from 3.6 to 6.3%; between-day CV at concentrations of 5 to 20 mg/L varied from 6.5 to 9.8%. Analytical recovery of piroxicam at 20 mg/L was 88%. Several other drugs were analyzed but none interfered with the assay. Serum concentrations found in 28 patients on a 20-30 mg/day dose ranged from 1.5 to 15.2 mg/L.