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.     

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.     

Validation of a high-pressure liquid chromatography and fluorescence polarization immunoassay for the determination of methadone in canine plasma

Methadone is a synthetic opiate derivative that possesses analgesic activity. A modified fluorescence polarization immunoassay (FPIA) method and a high-pressure liquid chromatography (HPLC) method with UV detection were compared for measurement of concentrations of methadone in canine plasma following intravenous and oral methadone administration. The mean+/-SD for accuracy (deviation from actual concentration) and precision (coefficient of variation) when methadone-fortified canine plasma was evaluated with the FPIA method were 3.9+/-3.2% and 4.4+/-2.9%, respectively. The accuracy and precision of the HPLC method were 6.2+/-5.2% and 7.7+/-3.9%, respectively. The limit of quantification for the FPIA and HPLC methods were 25 and 20 ng/mL, respectively. The coefficient of determination (r) between FPIA and HPLC analysis was 0.94 when plasma from dogs dosed with methadone was evaluated. FPIA provides a rapid, sensitive, and specific measurement of methadone in canine plasma following oral and intravenous administration.     

Evaluation of fluorescence polarization immunoassay for quantitation of serum salicylates

A fluorescence polarization immunoassay (FPIA) for serum salicylates that has been developed for use with the Abbott TDx analyzer is evaluated with regard to precision, accuracy, and stability of the standard curve. The FPIA method is also compared with a well-established high performance liquid chromatography (HPLC) technique in a clinical laboratory environment. The FPIA demonstrates excellent precision, and the standard curve is sufficiently stable to perform reproducible measurements over a 29-day period without recalibration. Superior accuracy of the FPIA method is indicated for salicylate concentrations between 50 and 800 micrograms/ml by recovery studies and by favorable comparison with the reference method. The performance of the FPIA for salicylate concentrations between 0 and 50 micrograms/ml is somewhat less favorable and should be used with caution in this range. The present method is more appropriate than HPLC for the management of patients receiving chronic high doses of salicylates or in cases of acute salicylate overdose and is also more rapid.     

Establishment of norvancomycin fluorescence polarization immunoassay for therapeutic drug monitoring

To establish a rapid and simple fluorescence polarization immunoassay method for determination of norvancomycin serum concentration, we collected 300 serum samples from the patients receiving norvancomycin in the hospitals localized in Shanghai, China. The drug concentrations were measured by the established HPLC method and FPIA with vancomycin kit. A FPIA algorithm for the determination of norvancomycin concentration was established according to the correlation between the FPIA and HPLC results. The methods and algorithm were validated in another 70 clinical samples. HPLC determination showed a good linear correlation within the range of 0.5-100?mg?l(-1) of norvancomycin concentrations. The method was validated via extraction recovery, intra- and inter-day methodological recovery and stability of norvancomycin in serum. Correlation analysis between the measurements of HPLC and FPIA in 300 serum samples gave the linear regression equation: (concentration by HPLC)=0.760 × (concentration by FPIA)-0.577 (P<0.001, R(2)=0.982). An algorithm was derived from this correlation for measuring the serum norvancomycin concentrations with FPIA. When it was validated in additional 70 serum samples from patients, 'FPIA algorithm' showed good accuracy versus HPLC: 'FPIA algorithm'=0.93 (HPLC)+0.63, R(2)=0.962, and 94.3% of the results from FPIA algorithm fell within the range of -20%/+20% of HPLC. This algorithm developed in this study can be easily used for determination of norvancomycin using TDx analyzer with vancomycin kit indirectly. It may also be useful for norvancomycin therapeutic drug monitoring.     

Evaluation of a fluorescence polarization immunoassay procedure for quantitation of isepamicin, a new aminoglycoside antibiotic

A fluorescence polarization immunoassay (FPIA) procedure for measuring isepamicin, a new aminoglycoside antibiotic, was evaluated. The range of the assay studied was from 0.3-50 micrograms/ml and the calibration curve can be stored for at least 32 days. There was no interference from hemoglobin (up to 400 mg/dl) and bilirubin (up to 20 mg/dl). Within-run precision on each 20 replicates at 5, 15, and 25 micrograms/ml yielded a coefficient of variation (CV) of 1.44-2.52%; between-run precision on analyses at 5, 15, and 25 micrograms/ml over five periods yielded a CV of 1.26-1.59%. Cross-reactivities of the assay system with tobramycin and amikacin were null, but that with gentamicin, of which isepamicin is a derivative, was approximately 14-29%. Data obtained by microbiological assay and FPIA and by high-performance liquid chromatography and FPIA correlated significantly, with coefficients of correlation being 0.980 (n = 70) and 0.965 (n = 39), respectively. The FPIA offers a rapid, efficient, and accurate system for therapeutic monitoring of isepamicin plasma levels.     

Liquid chromatography tandem mass spectrometry assay for topiramate analysis in plasma and cerebrospinal fluid: validation and comparison with fluorescence-polarization immunoassay

SUMMARY: The authors report the development and validation of a liquid chromatography tandem mass spectrometry assay (LC/MS/MS assay) for the analysis of topiramate (2,3:4,5-bis-o-(-1-methyl)-beta-D-fructopyranose sulfamate) in plasma and cerebrospinal fluid (CSF). Comparison is made with the commercially available fluorescence-polarization immunoassay (FPIA). LC/MS/MS ASSAY: Using the internal standard, 1,2:3,4-bis-o-(1-methylethylidene-alpha-D-galactopyranose sulfamate), a structural isomer, the calibration curve in plasma was linear in the concentration range of 0.02-20.0 mg/L (r(2) = 0.9998). The coefficients of variation in plasma were < or = 3%, and the accuracy ranged from 100% to 101% in the therapeutically relevant concentration range of 0.4-16.0 mg/L. In CSF, the mean recovery was 98%, and there was linearity between the nominal and the estimated concentration in the range of 1.5-20.0 mg/L (r(2)= 0.9996). FPIA: The calibration curve was linear in the concentration interval of 1.6-24.3 mg/L (r(2) = 0.9994), and the mean recovery was 96%. Accuracy in plasma was 99- 104%, and precision was 3.2-6.0%. In CSF, there was linearity between the nominal concentration and the estimated concentration in the range of 1.5-20.0 mg/L (r(2) = 0.9995), and the mean recovery was 100%. COMPARISON BETWEEN FPIA AND LC/MS/MS: There was a high correlation between the FPIA and the LC/MS/MS assay (r(2) = 0.9965 in plasma and r(2) = 0.9996 in CSF, P < 0.001 for both). In plasma and CSF, the two methods showed equal results, evaluated as the ratio between the two methods (plasma: median ratio = 1.00; 95% confidence interval [CI], 0.98-1.02, paired-sample test, P = 0.79; and CSF: median ratio = 1.00, 95% CI, 0.99-1.02, paired-sample test, P = 0.75). The coefficient of variation on the ratios between the two methods had similar levels: 5% in plasma and 3% in CSF. CONCLUSION: The new LC/MS/MS assay has favorable characteristics, being highly precise and accurate. FPIA also proved precise and accurate, and there was a high agreement with the LC/MS/MS assay in plasma and CSF. Either method displayed sufficient precision and accuracy and may thus be implemented in daily routine.     

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.     

Determination of N-desmethylmethsuximide serum concentrations using enzyme-multiplied and fluorescence polarization immunoassays

N-Desmethylmethsuximide (NDM), the active metabolite of the antiepileptic agent methsuximide, has been analyzed by gas-liquid chromatography and high-performance liquid chromatography (HPLC) in the past. This study compares methods using two commercially available immunoassays for ethosuximide, the enzyme multiplied immunoassay technique (EMIT) and fluorescence polarization immunoassay (FPIA), with an HPLC method for the measurement of NDM concentrations in serum. Within-day precision studies, utilizing low therapeutic (15.0 mg/L) and toxic (45.0 mg/L) NDM concentrations (n = 20), resulted in coefficients of variation (CVs) of 4.6 and 4.2%, respectively, for EMIT and 5.4 and 3.2%, respectively, for FPIA. Day-to-day precision studies (n = 10) resulted in CVs of 7.6 and 5.5%, respectively, for EMIT and 3.5 and 2.4%, respectively, for FPIA. No interference was observed from toxic concentrations of acetaminophen, caffeine, carbamazepine, methsuximide, phenobarbital, phensuximide, phenytoin, primidone, salicylate, and valproic acid in the EMIT and FPIA procedures. There was good linear correlation between EMIT and HPLC NDM determinations of 50 patient samples (r = 0.970; y = 0.96 x + 0.03), and a similar correlation between FPIA and HPLC NDM determinations in 48 patient samples (r = 0.975; y = 0.91 x + 1.24). Using ethosuximide reagents, both EMIT and FPIA systems can be adapted to reliably measure NDM serum concentrations.     

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.     

Evaluation of a mouse monoclonal antibody for the determination of serum theophylline by fluorescence polarization immunoassay

Theophylline concentrations in spiked human serum and serum specimens obtained from patients with normal and impaired renal function were measured by fluorescence polarization immunoassay (FPIA) using a mouse monoclonal antitheophylline antibody. The interday coefficients of variation of the assay after 14 days were 4.2, 3.3, and 2.4% at serum theophylline concentrations in pooled human serum of 7.1, 12.2 and 26.9 mg/L. Theophylline concentrations determined by FPIA and high performance liquid chromatography (HPLC) were used to generate the following linear regression equations relating the corresponding theophylline concentrations measured by each method in serum specimens from 50 patients with normal renal function (FPIA = 1.00 HPLC + 1.01, r = 0.98) and 50 patients with end-stage renal disease (FPIA = 1.04 HPLC + 0.08, r = 0.99). In contrast to previous studies performed with a polyclonal antitheophylline antibody, the precision, accuracy, and specificity of the FPIA were adequate for analysis of theophylline in serum specimens obtained from patients with end-stage renal disease.     

Comparison of high pressure liquid chromatography and fluorescence polarization immunoassay methods in a theophylline pharmacokinetic study

High pressure liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) were compared in a theophylline pharmacokinetic study. Eight healthy subjects received single 600-mg oral doses of two different sustained-release theophylline formulations. Fourteen blood samples were collected over 57 h after each dose, and the serum was analyzed for theophylline using both HPLC and FPIA methods. In comparing the two formulations using HPLC, there was no statistical difference in the area under the curve (AUC), terminal rate constant (k), or time of peak. However, there was a 13% difference in peak theophylline concentration (p less than 0.05). The same statistical conclusions were made for all parameters when using FPIA. When comparing the kinetic parameters determined with each assay, the AUC was 12% greater and the k was 17% smaller with FPIA (p less than 0.05). Orthogonal regression of all serum theophylline concentrations showed that FPIA = 1.04 HPLC + 0.20; r = 0.987, p less than 0.001. Stratification of serum theophylline concentrations into different ranges showed that FPIA overestimated the HPLC results in each range, but the percentage of overestimation was greater at lower concentrations (p less than 0.05). The use of FPIA seems appropriate in comparative studies of theophylline pharmacokinetics; however, the calculated kinetic parameters may differ slightly from those obtained with HPLC.     

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.     

The TDx assay for cyclosporine and its metabolites in blood samples compared with HPLC and RIA methods

Two methodologies have been developed to monitor cyclosporine (CsA) therapy: high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). Recently, a fluorescence polarization immunoassay (FPIA) has also become commercially available for the assay of CsA and its metabolites. The authors compared the results obtained with a modified FPIA with those found with two RIAs which use a polyclonal antibody, in order to verify if the FPIA assay is suitable for routine measurements in blood samples. Moreover, the accuracy of the RIAs and of the modified FPIA was checked against the results obtained by an HPLC technique assumed as a reference assay. The FPIA assay for CsA in blood samples seems preferable to the RIAs; in fact, as far as specificity is concerned, the TDx assay is comparable to polyclonal RIAs, while the precision (both within- and between-laboratories) is significantly better. Moreover, the TDx method is easier and faster to perform (20 samples can be assayed in about 30 min, while 2-4 h are necessary with RIA), with fewer handling steps; the instrumentation is automated and the reagents are more stable and less hazardous than those used in RIA.     

Determination of SR 49059 in human plasma and urine by LC-APCI/MS/MS

SR 49 059 ((2S 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene-sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide) is an orally active non-peptide vasopressin V_1a antagonist. A sensitive, selective, and robust LC-MS/MS method was developed to determine the plasma and urine concentrations of SR 49 059 in support of clinical studies. Plasma samples were prepared based on a rapid extraction procedure using Chem Elut™ cartridges. The extracted samples were analyzed on a C₁₈ HPLC column interfaced with a Finnigan TSQ 700 mass spectrometer. Positive atmospheric chemical ionization (APCI) was employed as the ionization source. The analyte and its internal standard (²H₆-SR 49 059) were detected by use of multiple reaction monitoring (MRM) mode. The plasma matrix had a calibration range 0.2−20 ng ml⁻¹, with within and between run accuracy and precision both less than 10%. The chromatographic run time was approximately 3 min. Urine samples were prepared based on a simple dilution with water, followed by analysis under the same conditions as plasma. The calibration range for urine matrix was 20–5000 ng ml⁻¹, with within and between run accuracy and precision less than 11%. The method has been successfully applied to the clinical sample analysis. The plasma assay was also evaluated on a Finnigan TSQ 7000 mass spectrometer. The performance based on precision and accuracy was virtually identical to that on the TSQ 700, with the exception of linearity in calibration curve (the TSQ 700 was linear, the TSQ 7000 was quadratic).     

浸透限制液相色谱、毛细管电泳和荧光偏振免疫法测定人血中苯巴比妥浓度的比较

目的:比较浸透限制高效液相色谱法(RAM-HPLC)、荧光偏振免疫法(FPIA)和高效毛细管电泳法(HPCE)测定人血中苯巴比妥(PB)浓度的异同。方法:建立一种直接进样 RAM-HPLC 法测定血中 PB 的浓度,并与 FPIA 和 HPC E法相比较。结果:三种方法测定 PB 血样浓度的结果有良好的相关性,相关系数分别为0.979(RAM-HPLC 对 FPIA),0.985(HPCE 对 FPIA)和0.989(HPCE 对 RAM-HPLC)。FPIA 法测定 PB 血样的值略高于 RAM-HPLC 和 HPCE 法。结论:RAM-HPLC 优点是准确度高和省去了样品前处理;HPCE 是分辨率高和污染少;FPIA 是简单和快速。三种方法都可用于PB 的治疗药物监测。     

Pharmaceutical availability of betamethasone dipropionate and gentamicin sulfate from cream and ointment

Kinetics of drug release from both compared preparations occuring as a cream and ointment, was in vitro studied. A reversed-phase HPLC method was developed for the determination of betamethasone dipropionate in lipophylic bases. Analyses were performed using a PLRP column with a mobile phase of methanol-acetonitrile-water and ultraviolet detection at lambda = 254 nm. The calibration curve was constructed for concentration (.0-50.0 microg/ml. The method is simple, accurate and precise. For the determination of gentamicin sulfate the FPIA method was used.     

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.     

Comparison of a new high-performance liquid chromatography method with fluorescence polarization immunoassay for analysis of methotrexate.

A simple high-performance liquid chromatography (HPLC) method for the determination of methotrexate (MTX) in biological fluids is described. The assay is rapid, the time required for analysis is less than 30 min, and it is sensitive, up to 0.01 microgram/ml, which is three times below the toxic MTX concentration. Fifty plasma samples drawn from acute lymphocytic leukemia (ALL) patients were used to compare this method with that of fluorescence polarization immunoassay (FPIA). A good correlation (r = 0.979) was obtained between the results of the two analyses. FPIA constantly overestimates the concentration in samples collected during elimination and underestimates those collected during infusion. The difference between the means of the two methods was 29% and 13% for the elimination and infusion samples, respectively. The means of the peak height ratio of the metabolite to MTX in the HPLC chromatograms were 3.39 and 0.33 during elimination and infusion, respectively. The results therefore indicate that HPLC is more specific when tracing the washout of MTX concentration. Because of this specificity and simplicity, the method is recommended for therapeutic drug monitoring. The stability of MTX in human saliva was investigated in this study. MTX was found to be stable at room temperature and at -20 degrees C for a minimum of 3 h and 3 weeks, respectively.     

Comparison of bioassay, high-performance liquid chromatography, and fluorescence polarization immunoassay for quantitative determination of vancomycin in serum

This investigation was designed to compare three assay techniques, the traditional bioassay (agar diffusion), and two more recent techniques, high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA), for the determination of vancomycin concentrations in serum. One hundred clinical samples obtained from patients receiving vancomycin were assayed by each method. The results from each assay were compared using linear regression analysis. The resultant correlation coefficients were as follows: 0.9996 for the HPLC versus FPIA, 0.7773 for the FPIA versus bioassay, and 0.7779 for HPLC versus bioassay. The FPIA technique was the easiest and fastest of the three methods; FPIA and HPLC were the most accurate.