Incorporating protein precipitation to resolve hybrid IP-LC-MS assay interference for ultrasensitive quantification of intact therapeutic insulin dimer in human plasma.

For pharmacokinetics characterization of a therapeutic insulin dimer, an ultrasensitive plasma method was required due to the expected low circulating levels in humans. A bioanalytical strategy combining immunoprecipitation enrichment with liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of the intact protein offers the opportunity to resolve the analyte from endogenous and exogenous insulin and insulin analogs. Nonetheless, interference from complex background matrix was observed limiting reliable measurements at the low concentration range. A sample preparation approach incorporating protein precipitation and immunoprecipitation was developed and optimized to further reduce sample complexity prior to LC-MS/MS analysis. This approach enabled a deeper level of selectivity and presented a cleaner mass spectrometric detection that may otherwise be confounded. Sample preparation was automated to allow high throughput analysis. The method reached a limit of quantitation at 0.3 ng/mL (25 pM), and a linear dynamic range from 0.3 to 300 ng/mL. Results were highly reproducible, with intra-day and inter-day precision and bias below 11%. Furthermore, the organic solvent treatment involved in protein precipitation is expected to improve assay resistance to the bias introduced by endogenous protein binding such as that exerted by anti-drug antibodies. The method was successfully applied to support clinical pharmacokinetics studies. This approach may potentially be adapted to bioanalysis of low abundance proteins.

Volumetric absorptive microsampling (VAMS®) in therapeutic protein quantification by LC-MS/MS: Investigation of anticoagulant impact on assay performance and recommendations for best practices in method development.

Microsampling techniques have been employed as an alternative to traditional serum/plasma sampling because of their inherently proven and desirable advantages across the pharmaceutical industry. These include reduced animal usage in pre-clinical studies, as well as, permitting the collection of samples that would otherwise be inaccessible in clinical studies. The application of volumetric absorptive microsampling (VAMS®) technology, a second-generation dried microsampling method, coupled with LC-MS, has been extensively explored for small molecule drugs at various drug development stages. However, the potential of using VAMS technology and LC-MS analysis for biological therapeutic development has yet to be well-established. In this work, we describe the method development, validation, and a proof-of-concept non-human primate study of a LC-MS/MS method for VAMS utilized to obtain pharmacokinetic (PK) data for a therapeutic monoclonal antibody. A good correlation between VAMS data and data from conventional serum samples was established in rhesus monkeys and indicated the possibility of using of this novel sampling technology in clinical studies. However, during the initial clinical study, a significant difference in internal standard (IS) response between the patient fingerstick samples and the standard/QC samples was observed, which posed a question on the accuracy of the clinical results. A comprehensive investigation confirmed that the EDTA anticoagulant used in the standard/QC samples was the root cause of the observed anomalous IS responses. Special considerations and corresponding best practices during method development and validation are proposed to ensure early detection of potential issues and appropriate implementation of VAMS technology in clinical studies in the future.

Toward highly sensitive and reproducible LC-MS/MS analysis of MK-8591 phosphorylated anabolites in human peripheral blood mononuclear cells.

Aim: MK-8591 (EFdA), a novel anti-HIV nucleoside analog, is converted to mono-, di- and tri-phosphates (MK-8591-MP, MK-8591-DP and MK-8591-TP) intracellularly, among which MK-8591-TP is the active pharmacological form. An ultrasensitive LC-MS/MS assay was required to measure MK-8591-DP and MK-8591-TP levels in human peripheral blood mononuclear cells (PBMCs). Sensitivity and reproducibility were major bottlenecks in these analyses. Materials and methods: Human PBMCs were isolated from blood and lysed with 70/30 methanol/RPMI-1640. An LC-MS/MS method was developed to simultaneously quantify MK-8591-DP and MK-8581-TP in PBMC lysates. Results: Low flow LC and dimethyl sulfoxide mediated signal enhancement enabled an extreme sensitivity with limit of quantitation at 0.1 ng/ml. Assay accuracy was 92.5-106% and precision was 0.7-12.1% for a linear curve range of 0.1-40 ng/ml. Matrix variability and interference liability were comprehensively evaluated. Conclusion: Our study findings and steps taken in addressing clinical sample issues help understand and overcome the challenges facing intracellular nucleotide analog analysis.

Strategy for peptide quantification using LC-MS in regulated bioanalysis: case study with a glucose-responsive insulin.

AIM: Advances in technology have led to a shift for peptide quantification from traditional ligand-binding assays to LC-MS/MS-based analysis, which presents challenges, in other assay sensitivity, specificity and ruggedness, in addition to lacking of regulatory guidance, especially for the hybrid assay format. Methodology & results: This report communicates a strategy that has been employed in our laboratories for method development and assay validation, and exemplified in a case study of MK-2640, a glucose-responsive insulin, in multiple matrices. Intact MK-2640 was monitored, while immunoaffinity purification and SPE were used to support the rat/dog GLP and clinical studies, respectively. The rationale and considerations behind our approach, as well as the acceptance criteria applied to the assay validation are discussed.

Extractability-mediated stability bias and hematocrit impact: High extraction recovery is critical to feasibility of volumetric adsorptive microsampling (VAMS) in regulated bioanalysis.

Volumetric absorptive microsampling (VAMS), a new microsampling technique, was evaluated for its potential in supporting regulated bioanalysis. Our initial assessment with MK-0518 (raltegravir) using a direct extraction method resulted in 45-52% extraction recovery, significant hematocrit (Ht) related bias, and more importantly, unacceptable stability (>15% bias from nominal concentration) after 7-day storage. Our investigation suggested that the observed biases were not due to VAMS absorption, sampling techniques, lot-to-lot variability, matrix effect, and/or chemical stability of the compound, but rather the low extraction recovery. An effort to improve assay recovery led to a modified liquid-liquid extraction (LLE) method that demonstrated more consistent performance, minimal Ht impact (Ht ranged from 20 to 65%), and acceptable sample stability. The same strategy was successfully applied to another more hydrophilic model compound, MK-0431 (sitagliptin). These results suggest that the previously observed Ht effect and "instability" were in fact due to inconsistent extractability, and optimizing the extraction recovery to greater than 80% was critical to ensure VAMS performance. We recommend adding Ht-independent recovery as part of feasibility assessment to de-risk the long-term extractability-mediated stability bias before implementing VAMS in regulated bioanalysis.

Novel Application of the Two-Period Microtracer Approach to Determine Absolute Oral Bioavailability and Fraction Absorbed of Ertugliflozin.

Ertugliflozin, a sodium glucose cotransporter-2 inhibitor, is approved in the United States for treatment of type 2 diabetes mellitus. A novel two-period study design with 14 C microtracer dosing in each period was used to determine absolute oral bioavailability (F) and fraction absorbed (Fa ) of ertugliflozin. Eight healthy adult men received 100-µg i.v. 14 C-ertugliflozin (400 nCi) dose 1 h after a 15-mg oral unlabeled ertugliflozin dose (period 1), followed by 100 µg 14 C-ertugliflozin orally along with 15 mg oral unlabeled ertugliflozin (period 2). Unlabeled ertugliflozin plasma concentrations were determined using high-performance liquid-chromatography tandem mass spectrometry (HPLC-MS/MS). 14 C-ertugliflozin plasma concentrations were determined using HPLC-accelerator mass spectrometry (AMS) and 14 C urine concentrations were determined using AMS. F ((area under the curve (AUC)p.o. /14 C-AUCi.v. )*(14 C-Dosei.v. /Dosep.o. )) and Fa ((14 C_Total_Urinep.o. /14 C_Total_Urinei.v. )* (14 C-Dosei.v. /14 C-Dosep.o. )) were estimated. Estimates of F and Fa were 105% and 111%, respectively. Oral absorption of ertugliflozin was complete under fasted conditions and F was ∼100%. Ertugliflozin was well tolerated.

Insulin glargine and its two active metabolites: A sensitive (16pM) and robust simultaneous hybrid assay coupling immunoaffinity purification with LC-MS/MS to support biosimilar clinical studies.

MK-1293 is a newly approved follow-on/biosimilar insulin glargine for the treatment of Type 1 and Type 2 diabetics. To support pivotal clinical studies during biosimilar evaluation, a sensitive, specific and robust liquid chromatography and tandem mass spectrometry (LC-MS/MS) assay for the simultaneous quantification of glargine and its two active metabolites, M1 and M2 were developed. Strategies to overcome analytical challenges, so as to optimize assay sensitivity and improve ruggedness, were evolved, resulting in a fully validated LC-MS/MS method with a lower limit of quantification (LLOQ) at 0.1ng/mL (∼16pM, equivalent to ∼2.8µU/mL) for glargine, M1 and M2, respectively, using 0.5mL of human plasma. The assay employed hybrid methodology that combined immunoaffinity purification and reversed-phase chromatography followed by electrospray-MS/MS detection operated under positive ionization mode. Stable-isotope labeled 6[D10]Leu-glargine and 4[D10]Leu-M1 were used as internal standards. With a calibration range from 0.1 to 10ng/mL, the intra-run precision (n=5) and accuracy were <6.21%, and 96.9-102.1%, while the inter-run (n=5/run for 7days) precision and accuracy were <9.55% and 96.5-105.1%, respectively, for all 3 analytes. Matrix effect, recovery, analyte stability, and interferences from control matrix, potential concomitant medications and anti-drug antibody were assessed. The assay was fully automated and has been successfully used in support of biosimilar clinical studies. Greater than 94.3% of incurred sample reanalysis (ISR) results met acceptance criteria, demonstrating the robustness of the assay. The strategic considerations during method development and validation are discussed, and can be applied to quantification of other peptides, especially insulin analogs, in the future.

A device for dried blood microsampling in quantitative bioanalysis: overcoming the issues associated blood hematocrit.

AIMS: A cross-laboratory experiment has been performed on a novel dried blood sampler in order to investigate whether it overcomes issues associated with blood volume and hematocrit (HCT) that are observed when taking a subpunch from dried blood spot samples. MATERIALS & METHODS: An average blood volume of 10.6 µl was absorbed by the samplers across the different HCTs investigated (20-65%). RESULTS: No notable change of volume absorbed was noted across the HCT range. Furthermore, the variation in blood sample volumes across six different laboratories was within acceptable limits. CONCLUSION: The novel volumetric absorptive microsampling device has the potential to deliver the advantages of dried blood spot sampling while overcoming some of the issues associated with the technology.

Direct comparison of radioimmunoassay and LC-MS/MS for PK assessment of insulin glargine in clinical development.

BACKGROUND: A direct comparison of radioimmunoassay (RIA) and LC-MS/MS for insulin glargine quantification in human plasma is provided. RESULTS: Compared with the RIA, the LC-MS/MS assay exhibited comparable/improved sensitivity (LLOQ at 0.1 ng/ml [˜16.7 pM or 2.8 µU/ml] for glargine and its metabolites M1 and M2, respectively) and ruggedness. Most importantly, it demonstrated a superior specificity advantage against the interference from endogenous insulin, exogenous insulin analogs (e.g., Novolog(®), Humalog(®) or Levemir(®), routine treatment for diabetes mellitus) and potentially pre-existing anti-insulin antibodies in patient samples. The data obtained from diabetic patients suggested the LC-MS/MS assay substantially improved pharmacokinetic characterization of glargine. CONCLUSION: LC-MS/MS overcame common limitations of RIA, and provided critically needed specificity to support glargine clinical development, without sacrificing assay sensitivity and ruggedness.

Small molecule specific run acceptance, specific assay operation, and chromatographic run quality assessment: recommendation for best practices and harmonization from the global bioanalysis consortium harmonization teams.

Consensus practices and regulatory guidance for liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays of small molecules are more aligned globally than for any of the other bioanalytical techniques addressed by the Global Bioanalysis Consortium. The three Global Bioanalysis Consortium Harmonization Teams provide recommendations and best practices for areas not yet addressed fully by guidances and consensus for small molecule bioanalysis. Recommendations from all three teams are combined in this report for chromatographic run quality, validation, and sample analysis run acceptance.

Merck's perspective on the implementation of dried blood spot technology in clinical drug development - why, when and how.

This paper communicates Merck's thoughts on why, when and how to use dried blood spot (DBS) technology in a clinical setting, and provides a strategic approach, emphasizing the necessary steps, for successful clinical implementation of this microsampling technique. PK consideration based on relevant in vitro data, that is, blood-to-plasma ratio, hematocrit, plasma unbound fraction and/or blood cell partition, is suggested to be part of the decision tree on when to choose DBS as a surrogate matrix for PK analysis. A quick feasibility assessment addressing analytical challenges, including sensitivity, hematocrit impact and storage stability, needs to be evaluated before initiating DBS studies. Special attention should be paid to the clinical sample collection procedures to ensure data quality. Bridging studies are required to establish the correlation between plasma and DBS data to ensure that pooling of data from the various clinical studies can be used in population PK or PK/PD assessment. Seeking regulatory feedback and guidance on a case-by-case basis is recommended.

Evaluation of dried blood spot (DBS) technology versus plasma analysis for the determination of MK-1775 by HILIC-MS/MS in support of clinical studies.

The collection of human blood samples as dried blood spots (DBS) for the pharmacokinetic assessment of investigational drugs in clinical trials offers a number of advantages over conventional plasma sampling, namely, small sample volume, simplified sample handling, and cost-effective shipping and storage. The use of DBS coupled with liquid chromatography-tandem mass spectrometry analysis was evaluated for the quantification of MK-1775, a Wee-1 inhibitor under development as a chemo/radio-sensitizer for the treatment of cancer. The DBS method exhibited an assay performance comparable to that of the existing plasma assay, which is currently used in support of clinical studies. Both assays used the same linear dynamic range of 2-1,000 ng/mL, with a lower limit of quantification of 2 ng/mL. Based on the intra-day assay validation results, the accuracy of the DBS method ranged from 94.0 to 105.0%, with a coefficient of variation of <4.8%. The blood-to-plasma ratio calculated from the DBS data (blood concentrations) and the plasma data (plasma concentrations) was in good agreement with the one obtained from the in vitro assessment using conventional methodology. No significant hematocrit impact on the assay was observed as hematocrit ranged from 16 to 85%. The correlation between the measured MK-1775 concentrations in plasma and that determined in dried blood spots from oncology patients during the ongoing clinical study was discussed.

Ultrasensitive liquid chromatography-tandem mass spectrometric methodologies for quantification of five HIV-1 integrase inhibitors in plasma for a microdose clinical trial.

HIV-1 integrase strand transfer inhibitors are an important class of compounds targeted for the treatment of HIV-1 infection. Microdosing has emerged as an attractive tool to assist in drug candidate screening for clinical development, but necessitates extremely sensitive bioanalytical assays, typically in the pg/mL concentration range. Currently, accelerator mass spectrometry is the predominant tool for microdosing support, which requires a specialized facility and synthesis of radiolabeled compounds. There have been few studies attempted to comprehensively assess a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach in the context of microdosing applications. Herein, we describe the development of automated LC-MS/MS methods to quantify five integrase inhibitors in plasma with the limits of quantification at 1 pg/mL for raltegravir and 2 pg/mL for four proprietary compounds. The assays involved double extractions followed by UPLC coupled with negative ion electrospray MS/MS analysis. All methods were fully validated to the rigor of regulated bioanalysis requirements, with intraday precision between 1.20 and 14.1% and accuracy between 93.8 and 107% at the standard curve concentration range. These methods were successfully applied to a human microdose study and demonstrated to be accurate, reproducible, and cost-effective. Results of the study indicate that raltegravir displayed linear pharmacokinetics between a microdose and a pharmacologically active dose.

Quantitative determination of odanacatib in human plasma using liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry analysis.

Odanacatib (ODN, MK-0822) is an investigational drug under development for the treatment of osteoporosis. A quantitative LC/MS-MS methodology was developed and validated to determine ODN concentrations in human plasma, with a linear calibration range from 0.500 to 500ng/mL. Stable isotope (13)C(6)-labeled ODN was employed as the internal standard (IS). Sample preparation was based on liquid-liquid extraction of basified plasma with methyl t-butyl ether in a 96-well plate format. The extracted samples were analyzed on a liquid chromatography-tandem mass spectrometry system equipped with a turbo ion spray source. Chromatographic separation of the analyte and IS was achieved on a Phenomenex Luna C18 (50mm×2.0mm, 5µm) column. Ion pairs m/z 526→313 for the analyte and m/z 532→319 for the IS were monitored in positive ionization mode for MS detection. This methodology has been fully validated and proved to be rugged and reproducible. Intra- and inter-run variability was within 5.88%, with accuracy between 95.6 and 106% of the nominal concentrations. Analyte stability was evaluated under various sample preparation, analysis and storage conditions. This assay has been utilized to analyze human plasma samples obtained from phase I to III clinical trials.

Immunoaffinity purification using anti-PEG antibody followed by two-dimensional liquid chromatography/tandem mass spectrometry for the quantification of a PEGylated therapeutic peptide in human plasma.

Quantification of a PEGylated peptide in human plasma using LC-MS/MS to support clinical studies presented challenges in terms of assay sensitivity, selectivity, and ruggedness. To ensure specific recognition of PEGylated species, an immunoaffinity purification method (IAP) using anti-PEG antibody followed by two-dimensional (2D) LC-MS/MS was developed for MK-2662, an investigational peptide containing 38 amino acids with a 40 kDa branched PEG [poly(ethylene glycol)] at C-terminus. Biotinylated anti-PEG antibody, bound to streptavidin-coated magnetic beads, was used to capture MK-2662 and its stable-isotope-labeled internal standard from human plasma. After on-bead digestion with trypsin, the supernatant was injected on a 2D high-performance liquid chromatography (HPLC) system constructed with strong cation-exchange and reversed-phase columns, followed by MS/MS detection of the surrogate N(1-12)-mer of MK-2662 on an API5000. The assay ruggedness was improved by optimizing the trypsin digestion and sample storage conditions. The intraday validation, conducted in parallel with protein precipitation (PPT) assay, demonstrated 94.8-105.8% accuracy with <9.76% coefficient of variation (CV) for IAP, and 99.0-101.0% accuracy with <3.43% CV for PPT, over a dynamic range of 2-200 nM and 1-1000 nM, respectively. A cross comparison, performed using clinical samples, showed that the values obtained from IAP assay were about 15-30% lower than those from PPT method, which supports more specific PEG recognition provided by IAP.

Determination of sitagliptinin human plasma using protein precipitation and tandem mass spectrometry.

A simple offline LC-MS/MS method for the quantification of sitagliptin in human plasma is described. Samples are prepared using protein precipitation. Filtration of the supernatants through a Hybrid-SPE-PPT plate was found to be necessary to reduce ionization suppression caused by co-elution of phospholipids with sitagliptin. The sitagliptin and its stable isotope labeled internal standard (IS) were chromatographed under hydrophilic interaction chromatography conditions on a Waters Atlantis HILIC Silica column (2.1 mm x 50 mm, 3 microm) using a mobile phase of ACN/H(2)O (80/20, v/v) containing 10 mM NH(4)Ac (pH 4.7). The sample drying after protein precipitation due to high organic content in the sample is not necessary, because HILIC column was used. The analytes were detected with a tandem mass spectrometer employing a turbo ion spray (TIS) interface in positive ionization mode. The multiple reaction monitoring (MRM) transitions were m/z 408-->235 for sitagliptin and m/z 412-->239 for IS. The lower limit of quantitation (LLOQ) for this method is 1 ng/mL when 100 microL of plasma is processed. The linear calibration range is 1-1000 ng/mL for sitagliptin. Intra-day precision and accuracy were assessed based on the analysis of six sets of calibration standards prepared in six lots of human control plasma. Intra-day precision (RSD%, n=6) ranged from 1.2% to 6.1% and the intra-day accuracy ranged from 97.6% to 103% of nominal values.

Hydrophilic interaction chromatography/tandem mass spectrometry for the simultaneous determination of three polar non-structurally related compounds, imipenem, cilastatin and an investigational beta-lactamase inhibitor, MK-4698, in biological matrices.

A method coupling hydrophilic interaction chromatography (HILIC) with tandem mass spectrometry (MS/MS) has been developed for the simultaneous determination of three polar non-structurally related compounds--a carbapenem antibiotic, imipenem (IMP), a renal dehydropeptidase inhibitor, cilastatin (CIL), and an investigational beta-lactamase inhibitor, MK-4698 (BLI), in rat plasma, monkey plasma and mouse blood. The analytes were extracted through protein precipitation, chromatographed on a Waters Atlantis HILIC column, and detected on a Sciex API4000 mass spectrometer using a Turbo-Ion Spray ion source in positive ionization mode following multiple-reaction monitoring (MRM). The assay dynamic range was 0.1-100 microg/mL for IMP, CIL and BLI, respectively, using a total of 20-25 microL biologic samples, and the total HPLC/MS/MS run time was 4 min/injection. The assay was found to be sensitive, selective and reproducible. The challenges, namely, sample stability, blood sample processing, matrix effect in monkey study samples, and dilution re-assays for the limited mouse blood samples, are resolved and discussed. This technique allowed rapid analysis of polar compounds in biologic matrixes with satisfactory chromatographic retention and increased throughput.

Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry.

To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5-500 nM and 5-5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C18 column (50 mm x 0.5 mm, 50 microm), followed by HPLC separation on a FluoPhase RP column (100 mm x 2.1 mm, 5 microm) and MS/MS detection. The off-line SPE assay used Waters Oasis HLB microElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm x 2.1 mm, 5 microm) for LC-MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of < 2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.

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 microL supernatant is directly injected into the LC-MS/MS system where separation is performed on a FluoPhase RP (150 x 2.1mm, 5 microm) 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 microL 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.

Determination of a novel gamma-secretase inhibitor in human plasma and cerebrospinal fluid using automated 96 well solid phase extraction and liquid chromatography/tandem mass spectrometry.

A method for determination of a gamma-secretase inhibitor, cis-3-[4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]propanoic acid (A), in human plasma and cerebrospinal fluid (CSF) has been developed to support the clinical investigation of compound A for its potential treatment of Alzheimer's disease. The method is based on HPLC with atmospheric pressure chemical ionization tandem mass spectrometric detection (APCI-MS/MS) in the negative ionization mode using a heated nebulizer interface. The addition of phosphoric acid at the ratio of 10-30microL per milliliter of human plasma or CSF was required during clinical sample collection to stabilize an acylglucuronide metabolite (C), which was potentially present in human plasma and CSF. Tween 20 (10% solution) was added at the ratio of 20microL per milliliter of CSF during CSF sample collection to prevent the loss of compound A during the storage of clinical samples. The compound A and its analog internal standard (B) in treated plasma or CSF were isolated from human plasma or CSF using solid phase extraction (SPE) in the 96 well format. The isolated analyte and internal standard were chromatographed on a Phenomenex Synergi Polar RP analytical column (50mmx3.0mm, 4microm), using a mobile phase consisting of 60/40 (v/v, %) acetonitrile/water at a flow-rate of 0.5mL/min. Tandem mass spectrometric detection was performed using a Sciex API 3000 tandem mass spectrometer operated in the multiple reaction monitoring (MRM) mode using precursor to product ion transitions of 441-->175 for A and 469-->175 for B, respectively. The assays were validated over the concentration range of 0.5-200ng/mL for human plasma and CSF. Replicate analyses (n=5) of spiked standards for both assays yielded a linear response with coefficients of variation less than 7% and accuracy within 5% of the nominal concentrations. In addition, the assays were automated to improve sample throughput by utilizing a Packard Multi PROBEII automated liquid handling system and a Tom-Tec Quadra 96 system. Numerous clinical studies have been supported using these assays.