LC-MS/MS Method for Determination of Teriflunomide, Over a 40,000-Fold Dynamic Range Using Overlapping Calibrators.

BACKGROUND: A liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been validated for use in therapeutic monitoring of the drug leflunomide in human serum and plasma. Because of concerns of teratogenicity, it is recommended that women who want to become pregnant have concentrations below 0.02 mcg/mL, although therapeutic levels are generally greater than 20 mcg/mL. Consequently, the method required a 40,000-fold dynamic range, which was achieved by dividing the curve range into 2 separate regions but with a single extraction procedure used for both. METHODS: A chromatographic separation was achieved between the parent drug and the active metabolite, teriflunomide (A77 1726), and the latter was quantified across a quantitative range of 0.005-200 mcg/mL. Samples were evaluated in an upper curve region first, with dilution, to determine whether the drug concentrations were in an appropriate therapeutic range. Samples that fell below the upper region were then reevaluated in the lower region without dilution. RESULTS: The method was shown to be reliable, with good accuracy and precision statistics, and acceptable quantitation using 4 different collection tube types. Mean accuracy over 6 control concentrations was within 5.4%, over 5 validation runs, whereas %coefficient of variation (CV) was within 8.15%. Evaluation of sodium heparin, KEDTA, NaF/K oxalate, and plain serum tubes from 6 separate individuals at the lower limit of quantification (LLOQ) showed no influence on the ability to quantify teriflunomide accurately. Regression equations for a curve range of 0.005-1 mcg/mL gave R values of 0.998 or better, whereas the range 0.8-200 mcg/mL had R values of 0.997 or better. CONCLUSIONS: The authors have developed and validated a method that allows quantification of leflunomide across a 40,000-fold range of 0.005-200 mcg/mL.

Correction for isotopic interferences between analyte and internal standard in quantitative mass spectrometry by a nonlinear calibration function.

Stable isotope-labeled internal standards are of great utility in providing accurate quantitation in mass spectrometry (MS). An implicit assumption has been that there is no "cross talk" between signals of the internal standard and the target analyte. In some cases, however, naturally occurring isotopes of the analyte do contribute to the signal of the internal standard. This phenomenon becomes more pronounced for isotopically rich compounds, such as those containing sulfur, chlorine, or bromine, higher molecular weight compounds, and those at high analyte/internal standard concentration ratio. This can create nonlinear calibration behavior that may bias quantitative results. Here, we propose the use of a nonlinear but more accurate fitting of data for these situations that incorporates one or two constants determined experimentally for each analyte/internal standard combination and an adjustable calibration parameter. This fitting provides more accurate quantitation in MS-based assays where contributions from analyte to stable labeled internal standard signal exist. It can also correct for the reverse situation where an analyte is present in the internal standard as an impurity. The practical utility of this approach is described, and by using experimental data, the approach is compared to alternative fits.

LC-MS/MS Method for the Quantification of the Leflunomide Metabolite, Teriflunomide, in Human Serum/Plasma.

Leflunomide is a prodrug that is metabolized to the active metabolite, teriflunomide (A77 1726), to inhibit the enzyme dihydroorotate dehydrogenase and decrease the synthesis of pyrimidine nucleotides for DNA and RNA synthesis. Teriflunomide is primarily used for the treatment of rheumatoid arthritis and multiple sclerosis.A liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated to quantify the drug teriflunomide over a concentration range of 5 ng/mL-200 µg/mL in serum or plasma. The calibration curve was divided into two separate overlapping regions of the analytical measurement range, with a high curve and a low curve range. Samples are first analyzed using the high-range calibration curve after a 100-fold dilution of the sample extract. Samples falling below the upper curve region are evaluated again without dilution and quantified, if possible, against the low curve calibration standards. This method can be used to support therapeutic drug monitoring of patients that are administered with leflunomide therapy.

Improving quantitative precision and throughput by reducing calibrator use in liquid chromatography-tandem mass spectrometry.

To improve efficiency in our mass spectrometry laboratories we have made efforts to reduce the number of calibration standards utilized for quantitation over time. We often analyze three or more batches of 96 samples per day, on a single instrument, for a number of assays. With a conventional calibration scheme at six concentration levels this amounts to more than 5000 calibration points per year. Modern LC-tandem mass spectrometric instrumentation is extremely rugged however, and isotopically labelled internal standards are widely available. This made us consider whether alternative calibration strategies could be utilized to reduce the number of calibration standards analyzed while still retaining high precision and accurate quantitation. Here we demonstrate how, by utilizing a single calibration point in each sample batch, and using the resulting response factor (RF) to update an existing, historical response factor (HRF), we are able to obtain improved precision over a conventional multipoint calibration approach, as judged by quality control samples. The laboratory component of this study was conducted with an existing LC tandem mass spectrometric method for three androgen analytes in our production laboratory. Using examples from both simulated and laboratory data we illustrate several aspects of our single point alternative calibration strategy and compare it with a conventional, multipoint calibration approach. We conclude that both the cost and burden of preparing multiple calibration standards with every batch of samples can be reduced while at the same time maintaining, or even improving, analytical quality.

Quantitation of Teriflunomide in Human Serum/Plasma Across a 40,000-Fold Concentration Range by LC/MS/MS.

Leflunomide is a prodrug used primarily for treatment of rheumatoid arthritis. The active metabolite, teriflunomide (A77 1726), inhibits the enzyme dihydroorotate dehydrogenase and thereby reduces the synthesis of pyrimidine ribonucleotides. Teriflunomide is also administered directly and finds use in treating multiple sclerosis. Therapeutic concentrations are generally in the tens of µg/mL serum or plasma and, due to adverse effects and the time required to reach steady state, therapeutic drug monitoring is beneficial. The drug is also a potential teratogen. A method was developed and validated to quantify the drug teriflunomide over a 40,000-fold concentration range of 5 ng/mL to 200 µg/mL in serum or plasma. This is accomplished by dividing the quantitative range into two separate but overlapping regions; a high curve and a low curve range. Samples are evaluated first against the high curve after a 100-fold dilution of the sample extract. Samples falling below the upper curve region are evaluated again without dilution and quantified, if possible, against the low curve calibration standards. Appropriate choice of a concentration for the deuterated internal standard (D4-teriflunomide) allows for a single, identical, extraction procedure to be performed for both curve regions but with the dilution performed for high curve samples. The method is rugged and reliable with good accuracy and precision statistics.

Validation and application of an automated 96 -well solid-phase extraction liquid chromatography-tandem mass spectrometry method for the quantitative determination of SCH 201781 in human plasma.

SCH 201781 is a direct thrombin inhibitor recently under study in clinical trials to determine its safety and efficacy for the treatment of venous and arterial thrombosis. In aqueous solution, SCH 201781 exists as three forms, a ring-opened hydrated form and two ring-closed diastereomers. An automated solid-phase extraction LC-MS/MS method that chromatographically separates and measures each form was developed and validated from 1 to 1000 ng/mL in human plasma. For calibration curve standards, within- and between-run precision (%CV) ranged from 0.6 to 13.7%, while accuracy (%bias) ranged from -4.8 to 13.1%. For quality control samples, within- and between-run %CV ranged from 1.5 to 9.9% while %bias ranged from -9.1 to 4.9%. The method requires a sample volume of 0.8 ml and utilizes 2H6-labeled SCH 201781 as the internal standard. For sample processing, an Isolute C-8 96-well solid phase extraction plate and a Tomtec Quadra 96 sample processor is employed. Separation of the three forms of SCH 201781 is achieved using a 5 microm, 2 mm x 100 mm Asahipak C8 HPLC column and gradient elution. A Sciex API 365 equipped with a turbo ionspray source is used in the selected reaction monitoring mode for detection. The validated method was used to support clinical studies.