Simultaneous determination of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone concentrations in rat serum by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry

An assay using high performance liquid chromatography (HPLC)-electrospray ionization-tandem mass spectrometry (ESI-MS-MS) was developed for simultaneously determining concentrations of morphine, oxycodone, morphine-3-glucuronide, and noroxycodone, in 50 mul samples of rat serum. Deuterated (d(3)) analogues of each compound were used as internal standards. Samples were treated with acetonitrile to precipitate plasma proteins: acetonitrile was removed from the supernatant by centrifugal evaporation before analysis. Limits of quantitation (ng/ml) and their between-day accuracy and precision (%deviation and %CV) were-morphine, 3.8 (4.3% and 7.6%); morphine-3-glucuronide, 5.0 (4.5% and 2.9%); oxycodone, 4.5 (0.4% and 9.3%); noroxycodone, 5.0 (8.5% and 4.6%). (C) 2004 Elsevier B.V. All rights reserved.

Measurement and stability of FTY720 in human whole blood by high-performance liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug FTY720, using HPLC-tandem mass spectrometry. Whole blood samples (500 mu l) were subjected to liquid-liquid extraction, in the presence of an internal standard (Y-32919). Mass spectrometric detection was by selected reaction monitoring with an atmospheric pressure chemical ionization source in positive ionization mode (FTY720: m/z 308.3 -> 255.3). The assay was linear from 0.2 to 25 mu g/l (r(2) > 0.997, n = 5). The inter- and intra-day analytical recovery and imprecision for quality control samples (0.5, 7 and 15 mu g/l) were 95.8-103.2 and < 5.5%, respectively. At the lower limit of quantification (0.2 mu g/l) the interand intra-day analytical recovery was 99.0-102.8% with imprecision of < 7.6% (n = 5). The assay had a mean relative recovery of 100.5 +/- 5.8% (n = 15). Extracted samples were stable for 16 h. IFTY720 quality control samples were stable at room temperature for 16 h at 4 degrees C for at least 8 days and when taken through at least three freeze-thaw cycles. In conclusion, the method described displays analytical performance characteristics that are suitable for pharmacokinetic studies in humans. (c) 2006 Elsevier B.V. All rights reserved.

Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry

High-performance liquid chromatography coupled by an electrospray ion source to a tandem mass spectrometer (HPLC-EST-MS/ MS) is the current analytical method of choice for quantitation of analytes in biological matrices. With HPLC-ESI-MS/MS having the characteristics of high selectivity, sensitivity, and throughput, this technology is being increasingly used in the clinical laboratory. An important issue to be addressed in method development, validation, and routine use of HPLC-ESI-MS/MS is matrix effects. Matrix effects are the alteration of ionization efficiency by the presence of coeluting substances. These effects are unseen in the chromatograrn but have deleterious impact on methods accuracy and sensitivity. The two common ways to assess matrix effects are either by the postextraction addition method or the postcolumn infusion method. To remove or minimize matrix effects, modification to the sample extraction methodology and improved chromatographic separation must be performed. These two parameters are linked together and form the basis of developing a successful and robust quantitative HPLC-EST-MS/MS method. Due to the heterogenous nature of the population being studied, the variability of a method must be assessed in samples taken from a variety of subjects. In this paper, the major aspects of matrix effects are discussed with an approach to address matrix effects during method validation proposed. (c) 2004 The Canadian Society of Clinical Chemists. All rights reserved.

The measurement of nicotine in human plasma by high-performance liquid chromatography-electrospray-tandem mass spectrometry

The authors describe a reverse-phase high-performance liquid chromatography-electrospray-tandem mass spectrometry method for the measurement of nicotine in human plasma. Samples (500 muL) with added deuterium-labeled d(3)-nicotine as an internal standard (IS) were treated with a 2-step process of ether extraction (6 mL) followed by back-extraction into 0.1% formic acid (50 muL). Chromatography was performed on a phenyl Novapak column with a mobile phase consisting of 50% 10 mM ammonium fortriate (pH 3.3) and acetonitrile (50:50, vol/vol). A flow rate of 0.2 mL/min resulted in a total analysis time of 5 minutes per sample. Mass spectrometric detection was by selected reactant monitoring (nicotine m/z 163.2 --> 130.2; IS m/z 166.2 --> 87.2). The assay was linear from 0.5 to 100 mug/L (r > 0.993, n = 9). The accuracy and imprecision of the method for quality control sampleswere 87.5% to 113% and < 10.2%, respectively. Interday accuracy and imprecision at the limit of quantification (0.5 mug/L) was 113% and 7.2% (n = 4). The process efficiency for nicotine in plasma was > 75%. The method described has good process efficiency, stabilized nicotine, avoided concentration steps, and most importantly minimized potential contamination. Further, we have established that water-based standards and controls are interchangeable with plasma-based samples. This method was used successfully to measure the pharmacokinetic profiles of subjects involved in the development of an aerosol inhalation drug delivery system.

Comparison of the pharmacokinetics of oxycodone and noroxycodone in male dark agouti and Sprague-Dawley rats: Influence of streptozotocin-induced diabetes

Purpose. The aims of this study are to evaluate whether cytochrome P450 (CYP)2D1/2D2-deficient dark agouti (DA) rats and/or CYP2D1/2D2-replete Sprague-Dawley (SD) rats are suitable preclinical models of the human, with respect to mirroring the very low plasma concentrations of metabolically derived oxymorphone seen in humans following oxycodone administration, and to examine the effects of streptozotocin-induced diabetes on the pharmacokinetics of oxycodone and its metabolites, noroxycodone and oxymorphone, in both rodent strains. Methods. High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry was used to quantify the serum concentrations of oxycodone, noroxycodone, and oxymorphone following subcutaneous administration of bolus doses of oxycodone (2 mg/kg) to groups of nondiabetic and diabetic rats. Results. The mean (+/- SEM) areas under the serum concentration vs. time curves for oxycodone and noroxycodone were significantly higher in DA relative to SD rats (diabetic, p < 0.05; nondiabetic, p < 0.005). Serum concentrations of oxymorphone were very low (< 6.9 nM). Conclusions. Both DA and SD rats are suitable rodent models to study oxycodone's pharmacology, as their systemic exposure to metabolically derived oxymorphone (potent mu-opioid agonist) is very low, mirroring that seen in humans following oxycodone administration. Systemic exposure to oxycodone and noroxycodone was consistently higher for DA than for SD rats showing that strain differences predominated over diabetes status.

A rapid HPLC-mass spectrometry cyclosporin method suitable for current monitoring practices

Objectives: Cyclosporin is an immunosuppressant drug with a narrow therapeutic window. Trough and 2-h post-dose blood samples are currently used for therapeutic drug monitoring in solid organ transplant recipients. The aim of the current study was to develop a rapid HPLC-tandem mass spectrometry (HPLC-MS) method for the measurement of cyclosporin in whole blood that was not only suitable for the clinical setting but also considered a reference method. Methods: Blood samples (50 mu L) were prepared by protein precipitation followed by C-18 solid-phase extraction while using d(12) cyclosporin as the internal standard. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface in positive ionization mode. Results: The assay was linear from 10 to 2000 mu g/L (r(2) > 0.996, n = 9). Inter-day,analytical recovery and imprecision using whole blood quality control samples at 10, 30, 400, 1500, and 2000 mu g/L were 94.9-103.5% and

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio similar to50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S2O3)(2)](3-) resulted in formation of the adducts HSA + Au(S2O3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S2O3)(2)](3-) blocked the formation of gold adducts. (C) 2003 Elsevier Inc. All rights reserved.