Optimisation of the sensitivity of an immunoassay analysis for tobramycin in serum

Tobramycin is an aminoglycoside antimicrobial drug frequently used in anti-pseudomonal therapy in cystic fibrosis and non-cystic fibrosis bronchiectasis patients. Therapeutic drug monitoring is routinely performed to increase efficacy and reduce the chance of toxicity. The most frequently used method to quantify tobramycin in serum or plasma is with an immunoassay method. However, immunoassays lack sensitivity to evaluate the lower concentrations of tobramycin for pharmacokinetic studies of for instance inhaled tobramycin. The aim of this study was to optimise the Syva® Emit® 2000 Tobramycin Assay combined with the ARCHITECT c8000. This adapted method was validated for accuracy and precision, having within-run, between-run variation. The adapted tobramycin immunoassay method has a linear range of 0.03 to 0.6 mg/L, which is comparable to liquid chromatography-mass spectrometry methods. The immunoassay method was validated with representative samples and has been implemented in routine analysis

Fast LC-MS/MS analysis of tacrolimus, sirolimus, everolimus and cyclosporin A in dried blood spots and the influence of the hematocrit and immunosuppressant concentration on recovery

We developed a method for the analysis of four immunosuppressants in dried blood spot (DBS) samples to facilitate therapeutic drug monitoring for transplant patients outside the hospital. An 8 mm disc from the central part of the DBS was punched, extracted and followed by LC-MS/MS analysis. The method was validated with ranges from 1.00-50.0 mu g/L for tacrolimus, sirolimus and everolimus, and from 20.0-2000 mu g/L for cyclosporin A. The validation showed a maximum overall bias of 13.0% for the sirolimus LLOQ while the maximum overall CV was 15.7% for the everolimus LLOQ. All four immunosuppressants showed to be stable in DBS for at least 7 days at 22 degrees C. The volume of the blood spot showed to have minor effect on measured concentrations. A cross-validation test between the 31 ET CHR paper and the Whatman FTA DMPK-C cards showed no significant difference between the two types of paper. During validation the hematocrit (HT) showed to have significant influence on the analytical results. When the measured concentrations were corrected for the effect of the HT, biases improved significantly. Additional recovery tests proved that the combination of especially low HT and high concentration does not only affect the spot size but can also affect the extraction recoveries of sirolimus and especially everolimus. Although the tested parameters like HT and concentrations are extreme and unlikely for routine analysis of outpatients, the fundamental effect of the combination of these parameters on extraction recoveries are proven with this research. The protein binding in the blood and hydrogen binding to the cellulose of the paper is suggested to influence extractions and gives new insights in the extraction methodology of DBS samples. The observed HT effect during the validation appeared to be negligible during the correlation study as no concentration corrections for the HT values were needed. Nevertheless, results from DBS samples with extremely high concentrations combined with extremely low HT values should be interpreted with caution. The patient correlation study showed good correlations with R-2 values higher than 0.87 between venous whole blood and venous DBS samples were observed for all four immunosuppressants. The Passing & Bablok plots showed positive biases of the slopes of 18% for tacrolimus and less than 12% for sirolimus, everolimus and cyclosporin A. The validated method, proved stability of the immunosuppressants in DBS, and the correlation study showed the capability of the DBS method to be used as an alternative for whole blood analysis in therapeutic drug monitoring. (c) 2013 Elsevier B.V. All rights reserved

Dried blood spot analysis of creatinine with LC-MS/MS in addition to immunosuppressants analysis

In order to monitor creatinine levels or to adjust the dosage of renally excreted or nephrotoxic drugs, the analysis of creatinine in dried blood spots (DBS) could be a useful addition to DBS analysis. We developed a LC-MS/MS method for the analysis of creatinine in the same DBS extract that was used for the analysis of tacrolimus, sirolimus, everolimus, and cyclosporine A in transplant patients with the use of Whatman FTA DMPK-C cards. The method was validated using three different strategies: a seven-point calibration curve using the intercept of the calibration to correct for the natural presence of creatinine in reference samples, a one-point calibration curve at an extremely high concentration in order to diminish the contribution of the natural presence of creatinine, and the use of creatinine-[H-2(3)] with an eight-point calibration curve. The validated range for creatinine was 120 to 480 mu mol/L (seven-point calibration curve), 116 to 7000 mu mol/L (1-point calibration curve), and 1.00 to 400.0 mu mol/L for creatinine-[H-2(3)] (eight-point calibration curve). The precision and accuracy results for all three validations showed a maximum CV of 14.0 % and a maximum bias of -5.9 %. Creatinine in DBS was found stable at ambient temperature and 32 A degrees C for 1 week and at -20 A degrees C for 29 weeks. Good correlations were observed between patient DBS samples and routine enzymatic plasma analysis and showed the capability of the DBS method to be used as an alternative for creatinine plasma measurement

Fast and Highly Selective LC-MS/MS Screening for THC and 16 Other Abused Drugs and Metabolites in Human Hair to Monitor Patients for Drug Abuse

Background:To facilitate the monitoring of drug abuse by patients, a method was developed and validated for the analysis of amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylphenidate, cocaine, benzoylecgonine, morphine, codeine, heroin, 6-monoacteylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), delta-9-tetrahydrocannabinol (THC), nicotine, and cotinine in human hair.Methods:The hair preparation method contains a 3-step wash procedure with dichloromethane followed by a simultaneous hair pulverization and extraction procedure with disposable metal balls. The developed liquid chromatography tandem mass spectrometry method uses a single injection to detect and confirm all 17 abused drugs, including THC, within 4.8 minutes.Results:Nicotine was validated with a linear range of 800-25,000 pg/mg hair, and all other substances were validated with a linear range of 30.0-2500 pg/mg hair. For inaccuracy and imprecision, the overall bias did not exceed -8.2% and the overall coefficient of variation did not exceed 17.7%. Autosampler stability was proven for 48 hours at 10 degrees C for all substances. Analytical cutoff concentrations were defined for each substance at the lowest validated inaccuracy and imprecision concentration with a bias and coefficient of variation within 15% and qualifier/quantifier ratios within 20% of the set ratio. The analytical cutoff concentrations were 200 pg/mg for codeine and 80.0 pg/mg for 6-MAM, heroin, EDDP, and THC. The analytical cutoff concentration for nicotine was 800 pg/mg and for all other validated substances 30.0 pg/mg. This method was successfully applied to analyze hair samples from patients who were monitored for drug abuse. Hair samples of 47 subjects (segmented into 129 samples) showed 3,4-methylenedioxymethamphetamine, methylphenidate, cocaine, benzoylecgonine, codeine, methadone, EDDP, THC, nicotine, and cotinine above the analytical cutoff.Conclusions:The method was fully validated, including the validation of the qualifier/quantifier ratios. The analysis of real hair samples proved the efficacy of the developed method for monitoring drug abuse. The results obtained by this method provide the physician or health-care professional with extensive information about actual drug abuse or relapse and can be used for patient-specific therapy

Determination of ribavirin in human serum using liquid chromatography tandem mass spectrometry

A method has been developed for the determination of ribavirin in human serum for therapeutic drug monitoring purposes, using liquid chromatography electrospray ionization mass spectrometry. Separation was obtained with a mobile phase gradient starting and ending in 100% aqueous conditions using a Waters Atlantis (R) T3 column (100 x 2 mm, 3 mu M). The entire sample preparation consisted of dilution, followed by ultrafiltration. From the clear ultrafiltrate 5 mu L was injected on the LC-MS/MS system. The calibration curves were linear in the range of 0.2-10 mg/L with within-run and between-run precisions (CVs) in the range of 0-10%. The method was validated with respect to specificity, selectivity, linearity, accuracy, precision, recovery and stability and meets the requirements of the FDA. The method was extensively tested for matrix effects by determining the variation of the slopes of calibration curves in different sources of serum and plasma. This method is suitable for the determination of ribavirin in human serum for therapeutic drug monitoring. (C) 2011 Elsevier B.V. All rights reserved

Application of Sweat Patch Screening for 16 Drugs and Metabolites Using a Fast and Highly Selective LC-MS/MS Method

Background: To facilitate the monitoring of drug abuse by patients, a method was developed and validated for fast and highly selective screening for amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, methylenedioxyamphetamine, methylenedioxyethylamphetamine, methylphenidate, cocaine, benzoylecgonine, morphine, codeine, heroin, 6-monoacteylmorphine, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, nicotine, and cotinine in PharmCheck sweat patches. The analysis of sweat patches would provide a noninvasive alternative matrix to urine or blood samples. Methods: The sweat patches were extracted during vigorous shaking for 10 minutes with 1.5 mL of 20 mmol/L ammonium formate, pH 7, and methanol (50%: 50% vol/vol). The extracts were cleaned up by filtering through Whatman mini-Uniprep syringeless filter vials before injection. The method uses a single injection to detect and confirm all 16 drugs and metabolites within 9.6 minutes. Results: The validated substances have a linear range of 3.0-300 nanograms per patch, except for nicotine which has a linear range of 30-3750 nanograms per patch. Stabilities of all substances in worn sweat patches were validated at room temperature for 7 days and as a processed sample in the autosampler at 10 degrees C for 5 days. Only heroin was unstable, with high individual variability and reported bias and coefficient of variation of, respectively, -30.6% and 22.1% in worn sweat patches at room temperature. The monitoring of ion ratios was added to the validation criteria. This resulted in analytical cutoff concentrations of 3.0 and 60 nanograms per patch for nicotine with validated qualifier/quantifier ratios. All analytical cutoff concentrations were lower than the cutoff concentrations proposed by the Substance Abuse and Mental Health Services Administration. Conclusions: The method uses validated cutoff concentrations, qualifier/quantifier ratios, and a simple extraction without extensive sample treatment for the analysis of 16 drugs and metabolites with a runtime of 9.6 minutes. This method was successfully applied for the analysis of 96 worn sweat patches to monitor patients for drug abuse. The results provided the physician or health-care professional with information about drug abuse and could be used to improve patient care with patient-specific therapy

Simultaneous determination of clarithromycin, rifampicin and their main metabolites in human plasma by liquid chromatography-tandem mass spectrometry

The drug combination rifampicin and clarithromycin is used in regimens for infections Caused by Mycobacteria. Rifampicin is a CYP3A4 inducer while clarithromycin is known to inhibit CYP3A4. During combined therapy rifampicin concentrations may increase and clarithromycin concentrations may decrease. Therefore a simple, rapid and easy method for the measurement of the blood concentrations of these drugs and their main metabolites (14-hydroxyclarithromycin and 25-desacetylrifampicin) is developed to evaluate the effect of the drug interaction. The method is based on the precipitation of proteins in human serum with precipitation reagent containing the internal standard (cyanoimipramine) and subsequently high-performance liquid chromatography (HPLC) analysis and tandem mass spectrometry (MS/MS) detection in an electron positive mode. The method validation included selectivity, linearity, accuracy, precision, dilution integrity, recovery and stability according to the "Guidance for Industry - Bioanalytical Method Validation" of the FDA. The calibration Curves were linear in the range of 0.10-10.0 mg/L for clarithromycin and 14-hydroxyclarithromycin and 0.20-5.0 mg/L for rifampicin and 25-desacetylrifampicin, with within-run and between-run precisions (CVs) in the range of 0% to - 10%. The components in human plasma are stable after freeze-thaw (three cycles), in the autosampler (3 days), in the refrigerator (3 days) and at room temperature (clarithromycin and 14-hydroxyclarithromycin: 3 days: rifampicin and 25-desacetylrifampicin: 1 day). The developed rapid and fully validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) method is Suitable for the determination of clarithromycin, 14-hydroxyclarithromycin, rifampicin and 25-desacetylrifampicin in human plasma. (C) 2009 Elsevier B.V. All rights reserved

Quantification of co-trimoxazole in serum and plasma using MS/MS

Background: Co-trimoxazole is frequently used in the prophylaxis and treatment of Pneumocystis carinii pneumonia. High plasma concentrations of sulfamethoxazole or trimethoprim are correlated with toxicity. There is, however, a large variation in PK observed which can lead to underexposure or toxicity. Results: We developed a novel LC-MS/MS method to analyze the components of co-trimoxazole, trimethoprim and sulfamethoxazole and its metabolite sulfamethoxazole-N-acetyl. This new method is expeditious due to its limited sample preprocessing and a relatively short run-time of only 3 min. Conclusion: This new method met the US FDA requirements on linearity, selectivity, precision, accuracy, matrix effects, recovery and stability and is suitable for routine analysis and future prospective studies

Quantification of amikacin and kanamycin in serum using a simple and validated LC-MS/MS method

Background: Amikacin and kanamycin are frequently used in the treatment of multidrug-resistant TB. The current commercially available immunoassay is unable to analyze kanamycin and trough levels of amikacin. The objective was therefore to develop a LC-MS/MS method for the quantification of amikacin and kanamycin in human serum. Materials & methods: Using apramycin as internal standard, selectivity, accuracy, precision, recovery, matrix effects and stability were evaluated. Results: The presented LC-MS/MS method meets the recommendations of the US FDA with a low LLOQ of 250 ng/ml for amikacin and 100 ng/ml for kanamycin. No statistical significant difference was found between the LC-MS/MS method and the immunoassay of amikacin (Architect((R)) assay, p = 0.501). Conclusion: The low LLOQ of amikacin and the ability to analyze kanamycin makes the LC-MS/MS method the preferred method for analyzing these aminoglycosides