Differential Mobility Spectrometry Coupled with Multiple Ion Monitoring in Regulated LC-MS/MS Bioanalysis of a Therapeutic Cyclic Peptide in Human Plasma.

A differential mobility spectrometry (DMS) in combination with a multiple ion monitoring (MIM) method was developed and validated for quantitative LC-MS/MS bioanalysis of pasireotide (SOM230) in human plasma. Pasireotide, a therapeutic cyclic peptide, exhibits poor collision-induced dissociation (CID) efficiency for multiple reaction monitoring (MRM) detection. Therefore, in an effort to increase the overall sensitivity of the assay, a DMS-MIM approach was explored. By selecting the most abundant doubly charged precursor ion in both the Q1 and Q3 of the mass analyzer in MIM and combining the DMS capability to significantly reduce the high matrix/chemical background noise, this new LC-DMS-MIM method overcomes the sensitivity challenge in the typical MRM method due to poor CID fragmentation of the analyte. Human plasma was spiked with pasireotide with concentrations in the range 0.01-50 ng/mL. Weak cation-exchange solid-phase extraction was employed for sample preparation. The sample extracts were analyzed with a SCIEX QTRAP 6500 system equipped with an ESI source and DMS device. The separation voltage and compensation voltage of the DMS and other parameters of the MS system were optimized to maximize signal responses. The performance of the LC-DMS-MIM assay for quantitative analysis of pasireotide in human plasma was evaluated and compared to those obtained via LC-MRM and LC-MIM without DMS. Overall, the assay sensitivity with DMS-MIM was approximately 5-fold better than that observed in MRM or MIM without DMS. The assay was validated with accuracy (% bias) and precision (% CV) of the QC results at eight concentration levels (0.01, 0.02, 0.05, 0.15, 0.3, 1.5, 15, and 37.5 ng/mL) evaluated ranging from -4.8 to 5.0% bias and 0.7 to 8.6% CV for the intraday and interday runs. The current LC-DMS-MIM workflow can be expanded to quantitative analysis of other molecules that have poor fragmentation efficiency in CID.

Practical approaches to incurred sample LC-MS/MS reanalysis: confirming unexpected results.

Incurred sample reanalysis (ISR) is an important step in assuring the quality of an LC-MS/MS bioanalytical assay and the integrity of bioanalysis conduct. A conventional ISR involves analysis of at least 20 samples taken from an in vivo study a second time using the method that was described in prestudy validation and employed in generating the initial study sample results. However, this practice is sometimes inadequate to confirm bioanalytical results that are unexpected. The present report discusses several additional exploratory activities that were performed to confirm the unexpected plasma concentration-time results of NVP-1, an investigational drug candidate, observed in the plasma samples collected from patients in a phase II trial. These approaches include (1) LC-MS/MS reanalysis of the study samples after multiple freeze/thaw cycles followed by a short-term benchtop storage, (2) evaluation of additional MS/MS transitions in LC-MS/MS, (3) employment of a different sample preparation procedure in LC-MS/MS, and (4) study sample dilution using plasma samples from healthy volunteers. These procedures are practical and can be readily implemented in the confirmatory LC-MS/MS bioanalysis of other small molecules.

Radiolabeled mass-balance excretion and metabolism studies in laboratory animals: a commentary on why they are still necessary.

The necessity of conducting traditional radiolabeled absorption, distribution, metabolism and excretion (ADME) studies in animals during development of new investigative agents has been questioned in a recent review. We present a compilation of the benefits of such studies in the understanding of the in vivo pharmacological activity and disposition of new drug candidates, including interpretation of preclinical toxicology findings, characterization of circulating metabolites, and determination of principal pathways of clearance. This understanding is valuable in anticipating the human disposition of the drugs and the planning of the clinical development program. Because of new technologies, evolving regulatory expectations, and increased scientific understanding of the disposition of drugs, the traditional design and timing of both animal and human ADME studies should be reviewed. Innovative "fit-for-purpose" studies may well be a better choice in a particular drug development program than a standard animal ADME "package". However, we submit that, at this time, radiolabeled animal ADME studies still provide a definitive and irreplaceable component of our understanding of the in vivo actions and behaviors of drugs and should continue to be performed prior to the exposure of large numbers of human subjects to investigative drugs.

Strategies in quantitative LC-MS/MS analysis of unstable small molecules in biological matrices.

Stability is an important pre-analytical variable for quantitative LC-MS/MS analysis of drug molecules and/or their metabolites in biological matrices. Instability of an analyte in any stage of the bioanalytical process, including sample collection, processing, storage, extraction and LC-MS/MS analysis, can result in under-/over-estimation if an adequate preventive procedure is not in place. In the current review on practical strategies in quantitative LC-MS/MS bioanalysis of unstable small molecules, the common causes of analyte instability were examined. The instability of some analytes is readily predictable because of the presence of certain chemically or biologically labile moieties in the molecules or because the compounds are in an inter-convertible form, e.g. lactone vs hydroxyl carboxylic acid. However, the instability of many other analytes is not readily predictable. Necessary evaluation needs to be conducted to identify the possible instability issues. The current review highlighted some general considerations and specific approaches for developing a robust LC-MS/MS method. In particular, incurred samples should be used as part of routine short-term stability assessment of any unstable analyte during the early stages of method development and validation. This can help unveil any 'hidden' instability issues that, if left unaddressed, could lead to the invalidation of a 'validated' method.

Bioanalysis in the Age of New Drug Modalities.

In the absence of regulatory guidelines for the bioanalysis of new drug modalities, many of which contain multiple functional domains, bioanalytical strategies have been carefully designed to characterize the intact drug and each functional domain in terms of quantity, functionality, biotransformation, and immunogenicity. The present review focuses on the bioanalytical challenges and considerations for RNA-based drugs, bispecific antibodies and multi-domain protein therapeutics, prodrugs, gene and cell therapies, and fusion proteins. Methods ranging from the conventional ligand binding assays and liquid chromatography-mass spectrometry assays to quantitative polymerase chain reaction or flow cytometry often used for oligonucleotides and cell and gene therapies are discussed. Best practices for method selection and validation are proposed as well as a future perspective to address the bioanalytical needs of complex modalities.

Dried blood spot sampling in combination with LC-MS/MS for quantitative analysis of small molecules.

The collection of whole blood samples on paper, known as dried blood spot (DBS), dates back to the early 1960s in newborn screening for inherited metabolic disorders. DBS offers a number of advantages over conventional blood collection. As a less invasive sampling method, DBS offers simpler sample collection and storage and easier transfer, with reduced infection risk of various pathogens, and requires a smaller blood volume. To date, DBS-LC-MS/MS has emerged as an important method for quantitative analysis of small molecules. Despite the increasing popularity of DBS-LC-MS/MS, the method has its limitations in assay sensitivity due to the small sample size. Sample quality is often a concern. Systematic assessment on the potential impact of various blood sample properties on accurate quantification of analyte of interest is necessary. Whereas most analytes may be stable on DBS, unstable compounds present another challenge for DBS as enzyme inhibitors cannot be conveniently mixed during sample collection. Improvements on the chemistry of DBS card are desirable. In addition to capturing many representative DBS-LS-MS/MS applications, this review highlights some important aspects of developing and validating a rugged DBS-LC-MS/MS method for quantitative analysis of small molecules along with DBS sample collection, processing and storage.

Supported liquid extraction in combination with LC-MS/MS for high-throughput quantitative analysis of hydrocortisone in mouse serum.

A high-throughput LC-MS/MS bioanalytical method was developed and validated for the determination of hydrocortisone in mouse serum via supported liquid extraction (SLE) in a 96-well plate format. Although sample extracts from SLE result in similar matrix effects compared with conventional liquid-liquid extraction (LLE), greater analyte extraction recovery and much higher analysis throughput for the quantitative analysis of hydrocortisone in mouse serum were obtained. The current LC-MS/MS method was validated for a concentration range of 2.00-2000 ng/mL for hydrocortisone using a 0.100 mL volume of mouse serum. The intra- and inter-day precision and accuracy of the quality control samples at low, medium and high concentration levels showed < or =12.9% CV and -3.4-6.2% bias for the analyte in mouse serum.

Juvenile toxicity assessment of d,l-methylphenidate in rats.

BACKGROUND: The oral administration of d,l-methylphenidate (MPH) was designed to encompass the major part of postnatal development in the rat and to evaluate potential chronic effects. METHODS: Wistar Hannover rats were cross-fostered on postpartum day 0 (day of birth) and were administered MPH at doses of 5, 50, and 100 mg/kg/day (mpkd) on postpartum days 7 to 70. Clinical signs, body weight, food consumption, developmental, behavioral, clinical/anatomic pathology, toxicokinetic, and fertility evaluations were conducted. RESULTS: MPH-related effects on clinical signs, body weight, and behavior tests were noted. Increased locomotor activity and cage biting/chewing occurred at > or =5 mpkd (females) and > or =50 mpkd (males) and were absent after dosing ceased. Body weight parameters were decreased at > or =50 mpkd and were comparable to controls at 5 weeks' recovery. Open field motor activity tests conducted 2 weeks after dosing ceased revealed decreased peripheral beam breaks at > or =50 mpkd. Passive avoidance tests conducted 3 weeks after dosing ceased indicated decreased females reaching learning criterion at 100 mpkd. This is considered of nominal significance as there were no effects in the water maze test or retention in passive avoidance test. After multiple doses, females exhibited higher exposures than males and exposures were reduced in all groups in comparison to those after a single dose. CONCLUSIONS: These results suggest that MPH can produce enduring behavioral effects in rats. The no-toxicologic-effect-level was 5 mpkd, associated with AUC((0-24 h)) racemate values in males and females, respectively, of 101 and 153 ng.h/mL after chronic dosing.

Developmental toxicity assessment of d,l-methylphenidate and d-methylphenidate in rats and rabbits.

BACKGROUND: Previous investigations reported no teratogenicity for methylphenidate (MPH). These studies investigated potential teratogenicity of d-MPH and d,l-MPH as commitments to the FDA. METHODS: Rabbits received 15, 50, 150 mg/kg/day (mkd) d-MPH or 20, 60, 200, 300 mkd d,l-MPH on gestation days 7-20. Rats received 2.5, 10, 40 mkd d-MPH, or 7, 25, 75, 80 mkd d,l-MPH on gestation days 6-17. RESULTS: d-MPH-In rabbits, mortality occurred at 150 mkd. Dilated pupils, increased activity, biting/chewing, respiration, and salivation occurred at >or=15 mkd in rabbits and >or=10 mkd in rats. Decreased food consumption occurred at 40 mkd in rats. Decreased body weight parameters occurred at 150 mkd in rabbits and >or=10 mkd in rats. There were no fetal findings in rabbits. In rats, skeletal variations occurred at 40 mkd. d,l-MPH-In rabbits, mortality occurred at >or=200 mkd. Dilated pupils, increased activity, biting/chewing, respiration, and salivation occurred at >or=20 mkd in rabbits and >or=25 mkd in rats. Decreased food consumption occurred at >or=200 mkd in rabbits and >or=25 mkd in rats. Decreased body weight parameters occurred at >or=200 mkd in rabbits and >or=25 mkd in rats. In rabbits, two fetuses (separate litters) had spina bifida and malrotated hindlimbs at 200 mkd. In rats, skeletal variations occurred at >or=75 mkd. CONCLUSIONS: There was no teratogenicity with d-MPH. There was a low teratogenic risk with d,l-MPH in only the rabbit. Higher C(max) may explain differences in results from previous studies.

Simultaneous determination of ribavirin and ribavirin base in monkey plasma by high performance liquid chromatography with tandem mass spectrometry.

For the first time, a liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the simultaneous determination of ribavirin and rabavirin base was developed and validated over the concentration range of 10-5,000 ng/ml, respectively, using a 0.025 ml monkey plasma sample. Ribavirin, ribavirin base, and the internal standards were extracted from monkey plasma via protein precipitation. After evaporation of the supernatant, the extract was reconstituted with 5% methanol (containing 0.1% formic acid) and injected onto the LC-MS/MS system. Optimum chromatographic separation was achieved on a Waters Atlantis dc18 (150 mm x 2.1mm, 5 microm) column with mobile phase run in gradient with 100% water containing 0.5% formic acid (A) and 90% acetonitrile (containing 0.5% formic acid (B). The flow rate was 0.4-0.6 ml/min with total cycle time of approximately 7.0 min. Post-column addition of acetonitrile (containing 0.1% formic acid) at 0.3 ml/min was used to increase the ionization efficiency in the MS source. The method was validated for sensitivity, linearity, reproducibility, stability and recovery. Lack of adverse matrix effect and carry-over was also demonstrated. The intra-day and inter-day precision and accuracy of the quality control (QC) samples were <9.0% relative standard deviation (R.S.D.) and 10.8% bias for ribavirin, and 10.3% R.S.D. and 11.3% bias for ribavirin base. The current specific, accurate and precise assay is useful in support of the toxicokinetic and pharmacokinetic studies of these compounds.

Quantitative analysis of pasireotide (SOM230), a cyclic peptide, in monkey plasma using liquid chromatography in combination with tandem mass spectrometry.

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of Pasireotide (SOM230) was developed and validated with a dynamic range of 0.5-250ng/ml using 50µl of monkey plasma. SOM230 and the internal standard, [M+6]SOM230, were extracted from monkey plasma via µElution SPE. The acidified sample matrix was loaded onto the preconditioned Waters SPE plate for further processing. The analyte was eluted from the SPE plate using freshly prepared elution solvent, which was followed by dilution and LC-MS/MS analysis. By eliminating a step of evaporation of the SPE eluent, instead, injecting the eluent after a simple dilution, compound loss due to non-specific binding to the 96-well materials was prevented. Furthermore, freshly prepared elution solution was found a key to optimal extraction recovery of the analyte from monkey plasma. The optimal chromatographic separation was achieved on an Atlantis dC18 (50×2.1mm, 5µm particle size) column using gradient elution with a total analysis cycle time approximately 4min per injection. The mobile phases were water containing 0.5% acetic acid and 0.05% trifluoroacetic acid (TFA) (mobile phase A) and acetonitrile containing 0.5% acetic acid and 0.05% TFA (mobile phase B). The incorporation of TFA (0.05%, v/v) and acetic acid (0.5%, v/v) into the mobile phases was accompanied by the improved chromatography and minimized carryover due to the HPLC column. The current method was validated for specificity, sensitivity, matrix effect, recovery, linearity, accuracy and precision, dilution integrity, batch size and stability. The accuracy and precision for the LLOQs (0.5ng/ml) were within ±5.6% bias and ≤7.8% CV, respectively. From the intra-day and inter-day evaluations, the precision of the other QC samples (1.5, 7.5, 75 and 190ng/ml) ranged from 2.7 to 4.9% CV and the accuracy (% bias) from -1.3 to 7.3%, respectively. Additional assessment of incurred sample reanalysis (ISR) was conducted to demonstrate the ruggedness and robustness of the assay method. The validated method was successfully implemented to support a toxicity study in monkeys administered with 5 and 30mg of SOM230 in a single intramuscular injection of a long acting release (LAR) formulation.

Quantitative analysis of factor P (Properdin) in monkey serum using immunoaffinity capturing in combination with LC-MS/MS.

AIM: Factor P (Properdin), an endogenous glycoprotein, plays a key role in innate immune defense. Its quantification is important for understanding the pharmacodynamics (PD) of drug candidate(s). RESULTS: In the present work, an immunoaffinity capturing LC-MS/MS method has been developed and validated for the first time for the quantification of factor P in monkey serum with a dynamic range of 125 to 25,000 ng/ml using the calibration standards and QCs prepared in factor P depleted monkey serum. The intra- and inter-run precision was ≤7.2% (CV) and accuracy within ±16.8% (%Bias) across all QC levels evaluated. Results of other evaluations (e.g., stability) all met the acceptance criteria. CONCLUSION: The validated method was robust and implemented in support of a preclinical PK/PD study.

High-throughput determination of ultra-low concentrations of LAG078, a lipid modulator, in human plasma.

A high throughput method with ultra-low level quantification limit (10 pg/ml) was developed and validated for the quantitative determination of LAG078, a lipid modulator, in human plasma to support clinical studies employing low doses of the compound. The method consisted of reverse phase chromatographic separation of the analyte from plasma extract followed by electrospray ionization (ESI) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring mode (MRM). Extraction was performed using a combination of protein precipitation and liquid-liquid extraction in the 96-well plate format to increase the throughput of the method. Optimised chromatographic separation in a short and high-resolution column (50 mm x 2.0 mm i.d., 3 microm particle size) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r=0.996) within the concentration range of 0.01 (lower limit of quantification) to 50 ng/ml using 0.5 ml of human plasma. The accuracy of the method varied from 95-101% with a precision (CV) of 5.29-13.2% over the concentration range. The method was simple and rapid.

Evaluation of plasma microsampling for dried plasma spots (DPS) in quantitative LC-MS/MS bioanalysis using ritonavir as a model compound.

Quantitative bioanalysis of dried plasma spots (DPS) is not subject to the impact of hematocrit and sample non-homogeneity that are often encountered in dried blood spot (DBS) assay. In the present report, an evaluation of plasma microsampling for DPS has been conducted for the first time using ritonavir as a model compound orally administered to dogs. For this evaluation, an LC-MS/MS method was developed and validated according to the current health authorities' guidance and industry practice for the analysis of ritonavir in DPS samples. The measured ritonavir concentrations in the DPS samples prepared using SAFE-TEC devices and directly from the conventional wet plasma using standard pipette were compared with each other and against those of conventional wet plasma. Both DPS results correlated well with each other and were comparable to those of the wet plasma. Good incurred sample reanalysis results were obtained for the two sets of DPS samples and wet plasma as well. The current plasma microsampling for DPS can serve as an alternative to DPS sampling via standard pipetting and wet plasma in in vivo studies.

LC-MS/MS bioanalysis of loratadine (Claritin) in dried blood spot (DBS) samples collected by subjects in a clinical research study.

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of loratadine, an H1 histamine antagonist, in human dried blood spot (DBS) samples following a single self-administered 10 or 20mg oral dose. The samples were produced by spotting approximately 30µl of whole blood onto PE-226 cards. Two 3-mm discs were cut from the DBS samples and extracted using aqueous methanol containing the internal standard. After transfer and drying of the resulting sample extract, the reconstituted residues were chromatographed using a Waters XSelect C18 column and isocratic elution for MS/MS detection. The possible impact due to hematocrit, volume of blood sample spotted, storage temperature, and humidity, on the accuracy of measured DBS results were investigated. The results showed that only spotted blood volume might have an impact; a small volume (10µl) tended to give a larger negative bias in the measured value than the large volume ones (≥20µl). The current method was fully validated over a dynamic range of 0.200-20.0ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.990. The intra-day accuracy and precision at the LLOQ were -11.5 to 0.0% bias and 6.4 to 8.9% CV, respectively. For the other QC samples (0.600, 3.00, 10.0 and 15.0ng/ml), the precision ranged from 4.2 to 9.8% CV and from 6.3 to 8.1% CV, respectively, in the intra-day and inter-day evaluations; the accuracy ranged from -1.7 to 10.0% and 2.7 to 5.3% bias, respectively, in the intra-day and inter-day batches. Loratadine is stable in the DBS samples for at least 271 days at ambient temperature in a desiccator, for at least 24h at 60°C and under 80% relative humidity, followed by re-conditioning at ambient temperature in a desiccator. The current methodology has been applied to determine the loratadine levels in DBS samples collected by subjects in a clinical research study to evaluate pharmacokinetic sampling in point-of-care setting.

Determination of LAG078, a lipid-lowering compound, in dog plasma using liquid chromatography-tandem mass spectrometry: application in a toxicokinetic study.

A high throughput method was developed and validated for the quantitative determination of LAG078, a lipid-lowering compound, in dog plasma obtained during toxicokinetic studies. The method was based on reverse phase liquid chromatographic separation of the analyte from plasma extract followed by turbo-ionspray (TIS) in the negative ion mode and tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. Extraction was performed using a combination of protein precipitation and liquid-liquid extraction in the 96-well plate format to increase the throughput of the method. Optimized chromatographic separation under basic condition (pH approximately 10) in a short polymer based column (50 mm x 2.0 mm i.d.) coupled with MRM mode of detection yielded clean chromatograms with minimal signal suppression. The standard curve was linear (r = 0.997) within the concentration range of 0.05 (lower limit of quantification; LLOQ) to 50 ng/ml using only 0.1 ml of dog plasma. The accuracy of the method varied from 95 to 100% with a precision (CV) of 3.04-10.8% over the concentration range. The method was simple, rapid, and robust.

A semi-automated LC-MS/MS method for the determination of LCI699, a steroid 11ß-hydroxylase inhibitor, in human plasma.

A novel liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of LCI699 was developed and validated with dynamic ranges of 0.0500-50.0 ng/mL and 1.00-1,000 ng/mL using 0.0500 mL and 0.100mL, respectively, of human plasma. LCI699 and the internal standard, [M+6]LCI699, were extracted from fortified human plasma via protein precipitation. After transfer or dilution of the supernatant followed by solvent evaporation and/or reconstitution, the extract was injected onto the LC-MS/MS system. Optimal chromatographic separation was achieved on an ACE C18 (50 mm × 4.6mm, 3 µm) column with 30% aqueous methanol (containing 0.5% acetic acid and 0.05% TFA) as the mobile phase run in isocratic at a flow rate of 1.0 mL/min. The total analysis cycle time is approximately 3.5 min per injection. The addition of an ion-pair reagent, TFA (0.05%, v/v), to the mobile phases significantly improved the chromatographic retention and resolution of the analyte on silica based reversed-phase column. Although addition of TFA to the mobile phase suppresses the ESI signals of the analyte due to its ion-pairing characteristics in the gas phase of MS source, this negative impact was effectively alleviated by adding 0.5% acetic acid to the mobile phase. The current method was validated for sensitivity, selectivity, linearity, reproducibility, stability and recovery. For the low curve range (0.0500-50.0 ng/mL), the accuracy and precision for the LLOQs (0.0500 ng/mL) were -13.0 to 2.0% bias and 3.4-19.2% CV, respectively. For other QC samples (0.100, 6.00, 20.0 and 40.0 ng/mL), the precision ranged from 1.2 to 9.0% and from 3.8 to 8.8% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -11.3 to 8.0% and -7.2 to 1.6% bias, respectively, in the intra-day and inter-day batches. For the high curve range (1.00-1,000 ng/mL), the accuracy and precision for the LLOQs (1.00 ng/mL) were 1.0-15.0% bias and 7.4-9.2% CV, respectively. For the other QC samples (3.00, 20.0, 200 and 750 ng/mL), the precision ranged from 0.8 to 7.0% and from 1.9 to 5.2% CV, respectively, in the intra-day and inter-day evaluations. The accuracy ranged from -2.5 to 4.0% and 0.7-1.0% bias, respectively, in the intra-day and inter-day batches. Additional assessments of incurred sample stability (ISS) and incurred sample reanalysis (ISR) were conducted to demonstrate the ruggedness and robustness of the assay method. The absence of adverse matrix effect and carryover was also demonstrated. The validated method was successfully used to support rapid turnaround human pharmacokinetic studies.

Developing a robust ultrafiltration-LC-MS/MS method for quantitative analysis of unbound vadimezan (ASA404) in human plasma.

Ultrafiltration of human plasma in combination with LC-MS/MS has been increasingly used in the quantitative analysis of the free fraction of drug candidates for PK/efficacy assessment. In addition to controlling the pre-incubation and centrifugation temperatures, some important factors that must be investigated and addressed include: (1) possible nonspecific binding, (2) possible impact of freeze/thaw cycles of plasma samples and extended storage of plasma samples at room temperature on the analyte recovery prior to ultrafiltration, and (3) identification of the appropriate assay dynamic range to avoid unnecessary dilutions. These factors were explored in the development and validation of a robust LC-MS/MS assay for the quantitative analysis of unbound vadimezan (ASA404) in human plasma. First, to mimic human physiological conditions, all plasma samples were incubated at ~37°C for a minimum of 30 min after thawing and prior to centrifugation to obtain the ultrafiltrate. Second, by passing the calibration standards and QC samples in plasma ultrafiltrate through the ultrafiltration membrane, the observed non-specific binding of the analyte due to the membrane was corrected. Third, the effects of multiple freeze/thaw cycles and/or storage at room temperature for various periods (4, 8, 16 and 24h) were evaluated to determine the impact on analyte concentrations in the ultrafiltrate from the plasma QC samples. Fourth, the appropriate dynamic range was established to accommodate the expected incurred sample free analyte concentrations. The validated assay has a dynamic range of 30.0-30,000 ng/ml for ASA404 in human plasma ultrafiltrate using a sample volume of 30 µl. Quality control pools containing the analyte were prepared at concentrations of 30.0-22,500 ng/ml to cover the assay calibration range. The intra-assay and inter-assay precision and accuracy were ≤ 15% (CV) and within ± 15% (bias) of the nominal values, respectively, for all measured QC concentrations, including the LLOQ. Freeze/thaw for up to three cycles of the plasma samples and/or the extended human plasma sample exposure to room temperature for up to 24h were confirmed to have no impact on the assay results for the free analyte. The validated method was successfully implemented to support clinical studies for the compound.

Quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spots using liquid chromatography-tandem mass spectrometry.

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 µl whole blood onto FTA cards. A 3mm disc was cut from the DBS samples and extracted using methanol, followed by liquid-liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C(18) column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0-5000 ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were -0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2-2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from -6.8 to 8.5% and -0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24h at room temperature and 4h at 60°C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤ -60°C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.

An investigation of incurred human urine sample reanalysis failure.

BACKGROUND: In this case study, urine samples were collected and transferred before the presence of a small degree of nonspecific binding was identified for the analyte of interest in human urine. The approach taken to address the issue was to use standards and quality controls to mimic the study samples and use Tween-80 (0.5%) to retrieve the adsorbed analyte. The method was validated, however, the incurred sample reanalysis (ISR) failed. RESULTS: Investigation into the ISR failure unveiled ineffective mixing of the study samples, with almost no headspace left inside the sample tubes after the addition of the surfactant using a regular vortex mixer, as the cause of the ISR failure. All samples were reanalyzed using a modified sample mixing method, which resulted in two successful ISR runs. CONCLUSIONS: Thorough sample mixing after the addition of surfactant is one of the important steps in ensuring accurate and reproducible analyses of urine samples with a small degree of analyte nonspecific binding.