Interferon-α 2b quantification in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC)

Interferon-α 2b (IFN-α 2b) is a recombinant therapeutic cytokine produced as inclusion bodies using a strain of Escherichia coli as expression system. After fermentation and recovery, it is necessary to know the amount of recombinant IFN-α 2b, in order to determine the yield and the load for solubilization, and chromatographic protein purification steps. The present work details the validation of a new short run-time and fast sample-preparation method to quantify IFN-α 2b in inclusion bodies using Reversed Phase-Ultra Performance Liquid Chromatography (RP-UPLC). The developed method demonstrated an accuracy of 100.28%; the relative standard deviations for method precision, repeatability and inter-day precision tests were found to be 0.57%, 1.54% and 1.83%, respectively. Linearity of the method was assessed in the range of concentrations from 0.05 mg/mL to 0.5 mg/mL, the curve obtained had a determination coefficient (r²) of 0.9989. Detection and quantification limits were found to be 0.008 mg/mL and 0.025 mg/mL, respectively. The method also demonstrated robustness for changes in column temperature, and specificity against host proteins and other recombinant protein expressed in the same E. coli strain.     

Simple GC-FID method development and validation for determination of α-tocopherol (vitamin E) in human plasma

This paper describes the development and validation of a novel GC-FID method for the determination of α-tocopherol concentration in human plasma which does not requires derivatization. The standard solutions and the plasma working solutions were prepared in absolute ethanol. To determine the concentration of α-tocopherol in human plasma, an aliquot of the plasma sample was deproteinized with ethanol. α-tocopherol was extracted with a mixture of hexane and dichloromethane (9:1). GC separation was performed using a HP-5 capillary column. Nitrogen was used as carrier gas at a flow-rate of 2 ml min^− 1. Calibration curves were linear over the concentration range 1–30 μg ml^− 1 (for standard solutions and solutions without endogenous α-tocopherol in plasma) and 5–34 μg ml^− 1 (for solutions with endogenous α-tocopherol in plasma). Absolute recovery, precision, sensitivity and accuracy assays were carried out. The analytical recovery of α-tocopherol from plasma averaged 97.44%. The limit of quantification (LOQ) and the limit of detection (LOD) of method for standard samples were 0.35 μg.ml^− 1 and 0.30 μg.ml^− 1, respectively. Within-day and between-day precision, expressed as the relative standard deviation (RSD) were less than 4%, and accuracy (relative error) was better than 8%. This novel method, developed and validated in our laboratory, could be successfully applied to the in-vivo determination of α-tocopherol. The endogenous α-tocopherol amounts in blood of twelve healthy volunteers with no vitamin drug usage were measured with this method.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

A method for CP 47, 497 a synthetic non-traditional cannabinoid in human urine using liquid chromatography tandem mass spectrometry

A rapid method has been developed to analyse CP 47, 497 in human urine. Urine samples were diluted with water:acetonitrile (90:10, v/v) and sample aliquots were analysed by triple quadrupole tandem mass spectrometry with a runtime of 5 min. Multiple reaction monitoring (MRM) as survey scan was performed. The method was validated in urine, according to an in-house validation protocol based on the criteria defined in Commission Decision 2002/657/EC. Three MRM transitions were monitored. The decision limit (CCα) was 0.01 μg mL^?1 and for the detection capability a (CCβ) value of 0.02 μg mL^?1 was obtained. The measurement uncertainty of the method was 21%. Fortifying human urine samples (n = 18) in three separate assays, show the accuracy of the method to be between 95 and 96%. The precision of the method, expressed as RSD values for the within-lab reproducibility at the three levels of fortification (0.1, 0.15 and 0.2 μg mL^?1) was less than 10% respectively. The method proved to be simple, robust and time efficient. To the best of our knowledge there are no LC–MS methods for the determination of CP 47, 497 with validation data in urine.     

Toxic and essential elements in children's blood (<6 years) from Kinshasa,DRC (the Democratic Republic of Congo)

In this study we determined the concentration of 9 trace elements (As, Cd, Cu, Hg, Mn, Mo, Pb, Se and Zn) in whole blood of children (n = 100, 64 girls, 36 boys and median age: 36 months) using inductively coupled plasma mass spectrometry (ICP-MS). The proportion of children potentially deficient in essential elements or poisoned by toxic elements was evaluated. The aging effects on the concentration of these elements were also investigated. The median values were 3.17 μg/L (As), 0.15 μg/L (Cd), 1.1 mg/L (Cu), 2.1 μg/L (Hg), 10.4 μg/L (Mn), 17.7 μg/L (Mo), 8.7 μg/dL (Pb), 10.7 μg/L (Se) and 5.0 mg/L (Zn). The concentration of many elements (As, Cd, Hg, Mn, Pb and Zn) showed significant age variations but not sex influence. Regarding levels of the essential elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except B-Cd, all toxic elements showed exceeded blood levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations.     

Ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) for the sensitive determination of folates in rice

High-performance liquid chromatography, coupled to tandem mass spectrometry (HPLC–MS/MS) has been established as the method of choice for the sensitive and simultaneous determination of different folates in a particular matrix, especially when only minute quantities of material are available. Using a previously developed and validated HPLC–MS/MS method as a starting point, we here report on the development and validation of an ultra-performance liquid chromatography (UPLC–MS/MS) method for analysis of folates in rice, which allows higher throughput and better resolution. UPLC was performed under gradient conditions on an Acquity HSS T3 column, followed by tandem mass spectrometry detection. The method was validated based on linearity, sensitivity, precision, accuracy and matrix effects. The limits of detection and the lower limits of quantification varied between 0.06 and 0.45 μg/100 g and 0.12 and 0.91 μg/100 g, respectively. Two linear calibration curves were established, one for the low and the other for the high concentration range. Analysis of the distribution and levels of folates in wild-type and folate-biofortified rice showed up to 50-fold enrichment in biofortified rice, with total folate levels of up to 900 μg/100 g rice. This is the first successful implementation of a UPLC method for the rapid and sensitive quantitative determination of folates in plant material.     

Quantification of raltegravir (MK0518) in human plasma by high-performance liquid chromatography with photodiode array detection

A precise and accurate high-performance liquid chromatography (HPLC) method with photodiode array detection has been developed and validated for raltegravir, a human immunodeficiency virus integrase strand transfer inhibitor (HIV-1 INSTI). Plasma (300 μL) was extracted with dichloromethane/hexane 50:50 (v/v) after addition of the internal standard, 6,7-dimethyl-2,3-di(2-pyridyl) quinoxaline. The compounds were separated using a dC18 column and detected with ultraviolet detection at 320 nm. The limit of quantification was 10 ng/mL for raltegravir. The method was linear and validated over a concentration range of 0–10,000 ng/mL. The intra-day precision ranged from 3.1 to 12.3%, while the intra-day accuracy ranged from ?15.0 to ?0.5%, the inter-day precision and accuracy were less than 7%. The mean recovery was 76.8%. Application to clinical samples taken from patients treated with raltegravir indicated that the method is suitable for measuring plasma concentrations of raltegravir in pharmacokinetic studies of clinical trials.     

Quantification of levetiracetam in plasma of neonates by ultra performance liquid chromatography–tandem mass spectrometry

A sensitive and specific method using ultra performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed for the determination of levetiracetam (LEV) in plasma of neonates. A plasma aliquot of 50 μl was deproteinized by addition of 500 μl methanol which contained 5 μg/ml UCB 17025 as an internal standard. After centrifugation, 50 μl of supernatant was diluted with 1000 μl of 0.1% formic acid–10 mM ammonium formate in water (pH 3.5) (mobile phase solution A) and 2 μl was injected onto the UPLC-system. Compounds were separated on a Acquity UPLC BEH C₁₈ 2.1 mm × 100 mm column using gradient elution with mobile phase solution A and 0.1% formic acid in methanol (mobile phase solution B) with a flow rate of 0.4 ml/min and a total runtime of 4.0 min. LEV and the internal standard were detected using positive ion electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The assay allowed quantification of LEV plasma concentrations in the range from 0.5 μg/ml to 150 μg/ml. Inter-assay inaccuracy was within ±2.7% and inter-assay precision was less than 4.5%. Matrix effects were minor: the recovery of LEV was between 97.7% and 100%. The developed method required minimal sample preparation and less plasma sample volume compared to earlier published LC–MS/MS methods. The method was successfully applied in a clinical pharmacokinetic study in which neonates received intravenous administrations of LEV for the treatment of neonatal seizures.     

Liquid chromatography tandem mass spectrometry method for the quantitation of NTBC (2-(nitro-4-trifluoromethylbenzoyl)1,3-cyclohexanedione) in plasma of tyrosinemia type 1 patients

In this study, we describe a bioanalytical method for quantification of NTBC in plasma of patients with hereditary tyrosinemia type 1 (HT-1) using high-performance liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). After protein precipitation with acetonitrile including Mesotrione as internal standard, separation of NTBC was achieved by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in selected reaction monitoring (SRM) mode. NTBC recovery in the developed method was found to be more than 90%. The lower limit of quantification was calculated to be 0.35 μM. The intra-day and inter-day precision of three different quality control samples (measured as RSD%) was less than 10% and 15%, respectively. The standard calibration curves showed good linearity within the range of 2.5–40 μM and the determined correlation coefficients were r² ≥ 0.995. This presented method is rapid, sensitive, specific and suitable for clinical practice and research.     

Validation of cell density and viability assays using Cedex automated cell counter

A method using Cedex automatic cell counter (Innovatis) to determine the cell density and viability of a whole cell-based immunotherapy product has been developed and validated for the assay performance characteristics including specificity, accuracy, precision, linearity, range, and robustness. Instrument-to-instrument variation due to intrinsic differences in handmade flow cells was also evaluated. For cell density, Cedex demonstrated acceptable specificity, accuracy and precision for cell densities ranging from 3.13 × 10⁵ to approximately 1.0 × 10⁷ cells/mL, with intermediate precision of about 5% relative standard deviation (RSD). However, a marked difference was observed between the two instruments studied and they therefore could not be used interchangeably without additional calibration procedures that went beyond the manufacturer's recommendation. For viability, mixing known numbers of non-viable cells with highly viable cells allowed evaluation of the specificity, accuracy and linearity of the viability determination. Acceptable levels of accuracy (95.3–106.4% recovery) and precision (RSD < 5%) were demonstrated for the viability range from 50 to 100%. The instrument-to-instrument difference was less than 4.6%. The assays for both cell density and viability were sufficiently robust for assay parameters. However, the effect of certain parameters was cell line-dependent, suggesting that Cedex performance should be verified for each cell line of interest.     

Evaluation of uncertainty of measurement from method validation data: An application to the simultaneous determination of retinol and α-tocopherol in human serum by HPLC

An isocratic RP-HPLC method for the determination of retinol and α-tocopherol in serum, with fluorescence and UV/VIS detection, respectively, was developed and validated according to international guidelines. Detection (retinol 0.015 mg/L, α-tocopherol 0.29 mg/L) and quantification (retinol 0.05 mg/L, α-tocopherol 0.95 mg/L) limits were determined. Repeatability was <3.3% and <2.9% and intermediate precision was <4.6% and <3.2%, for retinol and α-tocopherol, respectively. Certified reference materials were utilised to assess bias and guarantee traceability to SI units. Expanded uncertainties (retinol 8.9%; α-tocopherol 7.9%), estimated according to the EURACHEM/CITAC guide from method validation data, satisfied fit-for-purpose requirements based on biological variability.     

Determination of thiamin diphosphate in whole blood samples by high-performance liquid chromatography—A method suitable for pediatric diagnostics

An improved and easy to use method for the determination of thiamin diphosphate (TDP) in 100 μl of whole blood was developed. The small sample volume makes it possible to assess the nutritional status of vitamin B₁ in infants and even in preterm infants. Sample preparation comprises the extraction of TDP from whole blood by hemolysis, protein precipitation with trichloroacetic acid, and subsequent centrifugation. Potassium ferricyanide is used for pre-column derivatization of TDP to its fluorescent thiochrome derivative. Chromatographic separation was carried out using a reversed-phase column and an isocratic elution which consisted of a phosphate buffer and acetonitrile. TDP was detected fluorimetrically and quantified by external standardization. Method validation showed a high precision, almost complete recovery, and a high sensitivity. The lower limit of detection and the lower limit of quantification were 0.2 ng/ml and 4 ng/ml, respectively. Linearity was demonstrated over the expected concentration range of 4–400 ng/ml. In conclusion, we present a convenient HPLC method for the determination of TDP which is precise, sensitive and suitable for pediatric diagnostics.     

Validation of an optimized method for the determination of iodine in human breast milk by inductively coupled plasma mass spectrometry (ICPMS) after tetramethylammonium hydroxide extraction

In this study a novel method to determine iodine concentrations in human breast milk was developed and validated. The iodine was analyzed by inductively coupled plasma mass spectrometry (ICPMS) following tetramethylammonium hydroxide (TMAH) extraction at 90 °C in disposable polypropylene tubes. While similar approaches have been used previously, this method adopted a shorter extraction time (1 h vs. 3 h) and used antimony (Sb) as the internal standard, which exhibited greater stability in breast milk and milk powder matrices compared to tellurium (Te). Method validation included: defining iodine linearity up to 200 μg L⁻¹; confirming recovery of iodine from NIST 1549 milk powder. A recovery of 94–98% was also achieved for the NIST 1549 milk powder and human breast milk samples spiked with sodium iodide and thyroxine (T4) solutions. The method quantitation limit (MQL) for human breast milk was 1.6 μg L⁻¹. The intra-assay and inter-assay coefficient of variation for the breast milk samples and NIST powder were <1% and <3.5%, respectively. NIST 1549 milk powder, human breast milk samples and calibration standards spiked with the internal standard were all stable for at least 2.5 months after extraction. The results of the validation process confirmed that this newly developed method provides greater accuracy and precision in the assessment of iodine concentrations in human breast milk than previous methods and therefore offers a more reliable approach for assessing iodine concentrations in human breast milk.     

Development and validation of a method for the determination of a therapeutic peptide with affinity for the human B1 receptor in human plasma using HPLC-MS/MS

The peptide described in this report (MW 1180 Da; 10-amino acid synthetic peptide) is a potent and selective antagonist of the human B1 receptor (B1) that has been investigated for the treatment of chronic pain. A method to quantitate this peptide in human plasma has been developed to support human clinical trials designed to evaluate the safety, pharmacokinetics, and efficacy of this compound. Plasma samples (0.2 mL) were extracted using a Waters Oasis MAX (10 mg) 96-well plate and the resulting samples were analyzed using an Applied Biosystems API-5000 HPLC-MS/MS with an electrospray ionization (ESI) source. The method was validated for the determination of the B1 peptide in human plasma over the concentration range of 1–50 ng/mL. Isotopically labeled B1 peptide (¹³C6¹⁵N₂-B1 peptide) was used as an internal standard. Interday precision and accuracy, determined from analysis of quality control (QC) samples, yielded coefficients of variation (CV) of less than 5.3% and accuracy within a 2.4%. Within batch precision and accuracy determinations provided CV values of less than 7.3% and accuracy within a 6.0% bias. Precautions had to be taken to prevent B1 peptide loss to container surfaces and contamination of the HPLC-MS/MS. The validated assay was used in support of human clinical trials.     

Validation of a simple,rapid and cost effective method for the estimation of caprylic acid and sodium caprylate from biological products using NEFA-C kit

The method for the determination of caprylic acid and sodium caprylate from biological products was systematically validated using NEFA-C kit. The results obtained demonstrated that the kit method was simple, rapid, reliable, sensitive, reproducible and cost effective in comparison to the current methods i.e. colorimetric, High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) methods. The assay exhibited excellent linearity, accuracy, precision and robustness. Mean recoveries ranged between 95 and 101.3% (n = 6). The proposed method was linear over the concentration range of 0.05–10 mM of caprylate with values of coefficient of regression being >0.99. Method showed sensitivity of 0.05 mM (7.21 μg/ml for caprylic acid and 8.31 μg/ml for sodium caprylate). The % Relative standard Deviation (%RSD) for intra and interprecision studies was less than 5%. In conclusion the validated method was successfully used in monitoring of processed bulk and final products generated during production of biological products thus laying emphasis on strict control of release criteria for biological products fractionated using caprylic acid.     

A flexible and high throughput liquid chromatography–tandem mass spectrometric assay for the quantitation of telcagepant in human plasma

Telcagepant (MK-0974) is a novel oral calcitonin gene-related peptide (CGRP) receptor antagonist and is currently under clinical development. Results from phases II and III clinical trials have suggested that telcagepant is effective for migraine treatment. A reliable and high throughput protein precipitation (PPT) method for determination of telcagepant in human plasma using liquid chromatography coupled with atmospheric pressure chemical ionization (APCI) tandem mass spectrometry has been developed. Clinical samples, internal standard (IS) and acetonitrile are transferred into 96-well plates using a robotic liquid handling system. An aliquot of 10 μL supernatant is directly injected into the LC–MS/MS system where separation is performed on a FluoPhase RP (150 × 2.1 mm, 5 μm) column with an isocratic mobile phase (60% acetonitrile with 0.1% formic acid and 40% water with 0.1% formic acid) at 0.2 mL/min. The interfering 3S-diastereomer of telcagepant, which is observed in clinical samples, is chromatographically resolved from telcagepant. The PPT procedure significantly reduces the time required for sample processing and the assay is sufficiently sensitive for detection using both API 4000 and API 3000 mass spectrometers. The linear calibration range is 5–5000 nM using 200 μL of plasma. Assay intraday validation was conducted using six calibration curves derived from six lots of human control plasma. Calibration standard accuracy did not deviate by more than 3% and 6% of nominal values, and precision did not exceed 4% coefficient of variation (CV) and 10% CV, respectively on the API 4000 and API 3000. Several clinical phases IIb and III studies have been successfully supported with this assay.     

UHPLC method for the simultaneous determination of β-blockers, isoflavones and their metabolites in human urine

A rapid-resolution ultra high-performance liquid chromatography separation method (UHPLC) for the simultaneous determination of the following β-blockers: milrinone, sotalol, metoprolol, propranolol and carvedilol, and their metabolites: 5′-hydroxylphenyl-carvedilol, O-desmethylcarvedilol, 4-hydroxypropranolol, α-hydroxy-metoprolol, O-desmethyl-metoprolol; the following isoflavones: genistein, daidzein, glycitin, glycitein, puerarin and biochanin A; as well as their metabolites: dihydrogenistein, desmethylglycitein, 8-hydroxygenistein, daidzein-7,4′-diglucoside, 8-hydroxydaidzein, dihydrobiochanin A in human urine was optimized. The analysed compounds were extracted from human urine by means of solid phase extraction (SPE). The effective UHPLC separation of the examined compounds was applied on a Hypersil GOLD? (50 mm × 2.1 mm, 1.9 μm) column with a gradient mobile phase system and a UV detector. The complete separation of all analytes was achieved within 8.0 min. The method was validated for the determination of the aforementioned substances in human urine. The linear ranges, limits of detection (LOD) and limits of quantification (LOQ) for β-blockers, isoflavones and their metabolites were determined. The intra- and inter-day precision (%C.V.) was less than 4.48%, and the intra-day and inter-day accuracy was less than 4.74%. The tested SPE sorbent proved that appropriate absolute recoveries can be obtained for Oasis HLB (Waters). The mean recovery of the analytes, using the new SPE procedure, amounted from 70.14% to 99.85%. The present paper reports, for the first time, the method for the determination of β-blockers, isoflavones and their metabolites in human urine samples. The newly developed method was suitably validated and successfully applied for the analysis of the certain of the aforementioned analytes in human urine samples obtained from the patients suffering cardiovascular disease.     

Analysis methods and reference concentrations of 12 minor and trace elements in fish blood plasma

A comprehensive review of the analytical literature revealed substantial under-representation of trace element concentrations in fish blood, particularly for marine species. We describe a simple dilution procedure to measure Li, Mg, K, Ca, Mn, Cu, Zn, Se, Rb, Sr, Ba and Pb concentrations in low volumes of blood plasma of adult plaice (Pleuronectes platessa) using high resolution-inductively coupled plasma-mass spectrometry (HR-ICP-MS). Captive male and female plaice (n = 18) were serially sampled for one year and samples collected outside of the spawning season (n = 157) used to estimate reference ranges for this species. Method accuracy was deemed satisfactory, based on its application to the analysis of a certified reference material. Precision was generally <3%, with the most conservative measure of precision being ≤10% for all elements except Pb (～20%). This is the first study to analyse fish blood plasma by ICP-MS and includes some of the first reference ranges for trace element concentrations in fish blood.     

A sensitive LC–MS/MS method for quantification of a nucleoside analog in plasma: Application to in vivo rat pharmacokinetic studies

A LC–MS/MS method was developed and validated for determination of nucleoside analog (NA) in rat plasma. The method run time was 6 min and the limit of quantification (LOQ) was estimated at 100 pg/mL. The extraction procedure consisted on plasma samples protein precipitation with an acetonitrile solution which contained the stable isotope labeled internal standard (IS). Chromatography was performed on a newly developed C₁₆ column (150 mm × 4.6 mm, 5 μm) in order to avoid the use ion pair salts. The samples were eluted at 0.8 mL/min with a gradient of mobile phase made of water and acetonitrile both acidified with 0.5% acetic acid and 0.025% trifluoroacetic acid (TFA). A tandem mass spectrometer was used as a detector for quantitative analysis. Intra-run and inter-run precision and accuracy within ±15% were achieved during a 3-run validation for quality control samples at four concentration levels in rat plasma, over a concentration ranging between 0.1 and 1000 ng/mL. The data indicate that our LC–MS/MS assay is an effective method for the pharmacokinetics study of NA in rat plasma.     

Development and validation of a liquid chromatography/tandem mass spectrometry procedure for the quantification of sunitinib (SU11248) and its active metabolite, N-desethyl sunitinib (SU12662), in human plasma: application to an explorative study

A sensitive, precise and accurate quantitative liquid chromatography/tandem mass spectrometry (LC–MS/MS) method for the measurement of sunitinib (SU11248) and N-desethyl sunitinib (SU12662) in human plasma was developed and validated. All sample handling was done under strict light protection. The sample preparation method employed acetonitrile protein precipitation using d₅-SU11248 as an internal standard. The processed samples were chromatographed on a polymeric reversed-phase analytical column and analyzed by triple-quadrupole MS/MS in multiple reaction monitoring (MRM) mode using positive TurboIonSpray^® (TISP). The LC–MS/MS method described in this paper presents high absolute recovery (86.2% SU11248, 84.8% SU12662), high sensitivity (lower limit of quantitation of 0.06 ng/mL for both analytes), high inter-day precision (1.6–6.1% SU11248, 1.1–5.3% SU12662) and high analytical recovery (99.8–109.1% SU11248, 99.9–106.2% SU12662), as well as excellent linearity over the concentration range 0.060–100 ng/mL (r² > 0.999) with a short runtime of only 4.0 min. Results on the stability of SU11248 and SU12662 in human plasma are presented. During validation plasma from intensive care patients receiving many drugs were tested for interference and incurred samples were analyzed. The method met all criteria of the EMA and FDA guidelines during validation and was successfully applied to a pharmacokinetic study in healthy human volunteers.