First amyloid ß1-42 certified reference material for re-calibrating commercial immunoassays.

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .

Preanalytical approaches to improve recovery of amyloid-ß peptides from CSF as measured by immunological or mass spectrometry-based assays.

BACKGROUND: Amyloid-ß 1-42 (Aß1-42) peptide is a well-established cerebrospinal fluid (CSF) biomarker for Alzheimer's disease (AD). Reduced levels of Aß1-42 are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aß recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aß peptides in stored samples. METHODS: CSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aß1-42 concentrations were initially measured by immunoassay; subsequent determinations of CSF Aß1-42, Aß1-40, Aß1-38, Aß1-37, and Aß1-34 concentrations were made with an absolute quantitative mass spectrometry assay. In a second study, CSF from healthy control subjects and patients with dementia was denatured with guanidine hydrochloride (GuHCl) at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay. RESULTS: Two distinct immunoassays demonstrated that CSF Aß1-42 concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aß1-42 recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aß peptide mass spectrometry assay confirmed that Aß peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aß1-42. A comparison of CSF collection and processing methods suggested that Aß peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aß1-42/Aß1-40 ratio was a useful method to reduce variability. CONCLUSIONS: Analyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF Aß1-42 measurements. Sample denaturation during aliquoting increases recovery of Aß peptides and improves measurement accuracy. The Aß1-42/Aß1-40 ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.

CSF Aß1-42 - an excellent but complicated Alzheimer's biomarker - a route to standardisation.

The 42 amino acid form of amyloid ß (Aß1-42) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimer's disease. Several immunoassays for CSF Aß1-42 are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.g., clinical diagnostics) and selection of individuals for enrolment in clinical trials (patient stratification) remains challenging. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has initiated a working group for CSF proteins (WG-CSF) to facilitate standardisation of CSF Aß1-42 measurement results. The efforts of the IFCC WG-CSF include the development of certified reference materials (CRMs) and reference measurement procedures (RMPs) for key biomarkers. Two candidate RMPs for quantification of Aß1-42 in CSF based on liquid chromatography tandem mass spectrometry have been developed and tested in two ring trials. Furthermore, two commutability studies including native CSF pools, artificial CSF and spiked materials have been completed. On the basis of these studies, human CSF pools containing only endogenous Aß1-42 at three concentrations were selected as the format for future CRMs that are now being processed.

Development and validation of HILIC-ESI/MS/MS methods for simultaneous quantitation of several antipsychotics in human plasma and blood.

BACKGROUND: Knowledge of antipsychotic drug levels at point of care (POC) may significantly aid therapeutic decision-making. To support the development of future POC devices and to validate the use of fingerstick capillary blood sampling, two robust hydrophilic interaction LC-ESI/MS/MS methods were developed and validated. Two PK studies were completed evaluating the correlation between fingerstick blood and plasma concentrations with corresponding venous blood and plasma concentrations for several commonly prescribed atypical antipsychotics and selected metabolites. Sensitive and reliable LC-MS/MS bioanalytical assays were developed to support these studies. RESULTS: Three methods, requiring only 25-µl matrix volumes, were developed using supported liquid extraction with hydrophilic interaction LC-MS/MS detection and validated according to regulatory guidance. CONCLUSION: Robust and efficient LC-MS/MS assays were established and were effective in providing antipsychotic drug matrix comparator results in the intended clinical studies.

Comparison of a stable isotope labeled (SIL) peptide and an extended SIL peptide as internal standards to track digestion variability of an unstable signature peptide during quantification of a cancer biomarker, human osteopontin, from plasma using capillary microflow LC-MS/MS.

Human osteopontin (hOPN) is a secreted plasma protein which is elevated in various cancers and is indicative of poor prognosis. Here we describe investigations involving an extended peptide internal standard to track an unstable signature peptide followed by further method development and validation for quantitative measurement of hOPN from plasma using microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide 'GDSVVYGLR' was used as a signature peptide for this method. The optimized method involved immunocapture of the analyte protein using hOPN specific antibodies followed by trypsin digestion to obtain the signature peptide. Analysis was carried out on a Waters IonKey/MS system using a flow rate of 2.5µL/min. Immunocapture buffer was used as a surrogate matrix for the validation studies. The method was validated over a range of 25-600ng/mL. Intra-assay and inter-assay accuracies were within ±13%. Intra-assay and inter-assay precision were within 17%. A stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. Inherent digestion variability i.e., under controlled conditions, was within ±20% with both IS peptides. In digestion variability studies, where trypsin activity was varied (20-180%), the use of the extended SIL peptide as an internal standard limited the variability to within ±30% of the normalized response. Alternatively, when the SIL peptide was used as the internal standard, the variability ranged from -67.4% to 50.6% of the normalized response. The applicability of the validated method was demonstrated by quantification of OPN from plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. The plasma OPN concentrations in healthy individuals ranged from 38 to 85ng/mL with a mean concentration of 55.4±15.3ng/mL. A 1.5-12 fold increase in OPN concentrations, ranging from 85 to 637ng/mL, was seen in breast cancer patient samples.

An extended stable isotope-labeled signature peptide internal standard for tracking immunocapture of human plasma osteopontin for LC-MS/MS quantification.

A stable isotope-labeled signature peptide, whose sequence corresponds to the human osteopontin (hOPN) specific antibody epitope, was evaluated as an internal standard to compensate for immunocapture variability during quantification of hOPN by immunoaffinity-coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well from 150 to 4500 ng and analysis was carried out with internal standards added before and after the immunocapture step. The immunocapture variability ranged from -80.9 to 77.0% when the IS was added after immunocapture and from -37.5 to 20.3% when the internal standard was added before immunocapture. The lower variability demonstrates the ability of stable labeled isotope internal standard peptide to compensate for variation during immunocapture.

Development and optimization of on-line 2-dimensional chromatographic approaches for eliminating matrix effects and improving bioanalysis of peptides in human plasma using UHPLC-MS/MS.

Online 2-dimensional chromatographic approaches for eliminating matrix effects and optimizing bioanalysis of peptides using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were studied. Three therapeutic peptides (octreotide, desmopressin, and vasopressin) were selected as model analytes. Human plasma was precipitated with acetonitrile; peptides were analyzed on C(8), C(18), Phenyl and HILIC ACQUITY UPLC columns. For simpler online clean-up applications, a C(18) pre-column was coupled to the analytical column via a switching valve. For more complex heart-cutting applications, two analytical columns were used with optional online dilution to refocus the analyte peaks prior to the second dimension separation. This allows the use of MS incompatible mobile phases, such as TFA, in the first dimension separation. Online clean-up effectiveness was investigated by monitoring phospholipids. Flushing direction, mobile phase composition, flow rate and transfer window were evaluated. Phospholipids were readily retained on reversed-phase columns, and the peptides were reproducibly transferred, individually or as a group, to the second column using appropriate transfer windows. The best peak shapes were obtained when the second dimension column was more retentive (e.g. C(18) vs. C(8)). However, C(8) to HILIC gave broad unresolved peaks due to mobile phase mismatch. Trapped phospholipids were efficiently removed from either guard columns or first dimensional columns by forward- or back-flushing at high flows; however, back-flushing was more efficient with lower flow rates on larger columns.

Investigation of endogenous blood lipids components that contribute to matrix effects in dried blood spot samples by liquid chromatography-tandem mass spectrometry.

Dried blood spot (DBS) sampling coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a rapidly developing approach in the field of biopharmaceutical analysis. DBS sampling enables analysis of small sample volumes with high sensitivity and selectivity while providing a convenient easy to store and ship format. Lipid components that may be extracted during biological sample processing may result in matrix ionization effects and can significantly affect the precision and accuracy of the results. Glycerophosphocholines (GPChos), cholesterols and triacylglycerols (TAG) are the main lipid components that contribute to matrix effects in LC-MS/MS. Various organic solvents such as methanol, acetonitrile, methyl tertiary butyl ether, ethyl ether, dichloromethane and n-hexane were investigated for elution of these lipid components from DBS samples. Methanol extracts demonstrated the highest levels of GPChos whereas ethyl ether and n-hexane extracts contained less than 1.0 % of the GPChos levels in the methanol extracts. Ethyl ether extracts contained the highest levels of cholesterols and TAG in comparison to other investigated organic solvents. Acetonitrile is recommended as an elution solvent due to low lipid recoveries. Matrix effects resulted from different extracted lipid components should be studied and assessed carefully in DBS samples.

A randomized, multiple-dose parallel study to compare the pharmacokinetic parameters of synthetic conjugated estrogens, A, administered as oral tablet or vaginal cream.

OBJECTIVE: A randomized, parallel-design study was conducted to determine the pharmacokinetic profile of synthetic conjugated estrogens A (SCE-A) vaginal cream (0.625 mg SCE-A/g) when administered at intervals (1 g once daily for 7 d, then twice weekly) over a 27-day period as compared with the pharmacokinetic profile of 0.3 mg SCE-A tablets administered once daily orally for 27 days. METHODS: Blood samples were collected 48 hours before initial dosing for baseline levels and at multiple occasions during the study until 48 hours after final study dosing (day 29). Maximum plasma concentration, time to maximum plasma concentration (Tmax), and area under the curve from 0 to 24 hours were calculated at days 1, 7, and 27; in addition, area under the curve from 0 to 48 hours was calculated at days 7 and 27, and area under the curve weekly (AUCweekly) values were calculated for both groups. For purposes of comparison, ratios of AUCweekly values for vaginal cream and oral tablets were analyzed. RESULTS: Compared with an oral daily dose of 0.3 mg SCE-A, the steady-state systemic exposure from vaginal cream application was considerably less, with the cream-to-oral ratio being 0.45 for baseline-adjusted (BA) unconjugated estradiol, 0.30 for BA unconjugated estrone, and 0.04 for unconjugated equilin (AUCweekly). At steady-state, the systemic blood levels of BA unconjugated estrone, BA unconjugated estradiol and unconjugated equilin were significantly lower in women who received biweekly application of 1 gm vaginal cream compared to women who took an oral daily dose of 0.3 mg SCE-A tablet. CONCLUSIONS: After intravaginal application of SCE-A vaginal cream, absorption of estrogens was lower compared with absorption after oral administration. At steady state, the systemic exposure of equilin, estradiol, and estrone was significantly lower after twice-weekly administration of 1 g SCE-A vaginal cream compared with that achieved with an oral daily dose of a 0.3 mg SCE-A tablet.

Investigation of endogenous blood plasma phospholipids, cholesterol and glycerides that contribute to matrix effects in bioanalysis by liquid chromatography/mass spectrometry.

Matrix effects caused by compounds endogenous to the biological sample are a primary challenge in quantitative LC/MS/MS bioanalysis. Many approaches have been developed to minimize matrix effects such as optimization of sample extraction procedures and use of isotopically labeled internal standards. Unexpected matrix components may still remain undetected, however, because of the selective mass transitions monitored during MS/MS analysis. Glycerophosphocholines are the major phospholipids in plasma that have been widely shown to cause significant matrix effects on electrospray ionization efficiencies for target analytes. The purpose of this work was to investigate potential matrix effects resulting from different endogenous lipid classes, including phospholipids, acylglycerols and cholesterols, in order to establish a library for the relative presence of these components in biological sample extracts obtained by commonly used sample preparation techniques. Thirteen compounds were selected which were representatives of eight phospholipids classes, mono, di, triacylglycerols, cholesterol and cholesterol esters. Post-column infusion experiments were carried out to compare relative ion suppression effects of these compounds. Chlorpheniramine and loratadine were selected as model test analytes. A Concentration Normalized Suppression Factor (%CNSF) was defined to allow comparison of ion suppression effects resulting from different endogenous lipids according to their typical concentrations in human plasma and erythrocytes. A simple LC/MS/MS method was developed to monitor these endogenous components in sample extracts and their extraction recoveries from a plasma pool were compared using protein precipitation, liquid-liquid extraction, supported-liquid extraction, solid phase extraction and Hybrid SPE-precipitation methods. Endogenous lipid components other than GPChos, such as cholesterols and triacylglycerols, may result in significant matrix effects and should be monitored during method development. No single extraction procedure was efficient in removing all of the various lipid components. Use of the results presented here, along with a consideration of analyte chemical structure, the type of matrix and the type of sample preparation procedure, may help a bioanalytical scientist to better anticipate and minimize matrix effects in developing LC/MS/MS-based methods.

An improved LC-ESI-MS-MS method for simultaneous quantitation of rosiglitazone and N-desmethyl rosiglitazone in human plasma.

A fast, sensitive, and selective method for the simultaneous quantitation of rosiglitazone and N-desmethyl rosiglitazone in human plasma, using rosiglitazone-d(4) and N-desmethyl rosiglitazone-d(4) as the respective internal standards, has been developed and validated. The analytes in human plasma (50 microL sample aliquot) were isolated through supported liquid/liquid extraction (SLE) and separated by isocratic HPLC over a 3-min period. The precursor and product ions were detected by ESI-MS-MS with multiple reaction monitoring (MRM) in a triple quadrupole mass spectrometer. For both rosiglitazone and N-desmethyl rosiglitazone, the lower limit of quantitation (LLOQ) was 1.00 ng/mL, and the quantitation range was 1.00-500 ng/mL (with an average correlation coefficient >0.9990). The intra-assay and inter-assay precision had a maximum %CV of 9.37%, and the accuracy had a maximum %difference from theoretical of 12.7%. This method was applied to a clinical study where 16 healthy volunteers were administered a single dose of 4.0mg rosiglitazone. The pharmacokinetic parameters of rosiglitazone and N-desmethyl rosiglitazone were consistent with the results reported in the literature.

Separation of a BMS drug candidate and acyl glucuronide from seven glucuronide positional isomers in rat plasma via high-performance liquid chromatography with tandem mass spectrometric detection.

A high-performance liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of a BMS drug candidate and its acyl glucuronide (1-O-beta glucuronide) in rat plasma. A 50-microL aliquot of each plasma sample was fortified with acetonitrile containing the internal standard to precipitate proteins and extract the analytes of interest. After mixing and centrifugation, the supernatant from each sample was transferred to a 96-well plate and injected into an LC/MS/MS system. Chromatographic separation was achieved isocratically on a Phenomenex Luna C(18), 3 mm x 150 mm, 3 microm column. The mobile phase contained 0.075% formic acid in 70:30 (v/v) acetonitrile/water. Under the optimized chromatographic conditions, the BMS drug candidate and its acyl glucuronide were separated from its seven glucuronide positional isomers within 10 min. Resolution of the parent from all glucuronides and acyl glucuronide from its positional isomers was critical to avoid their interference with quantitation of parent or acyl glucuronide. Detection was by positive ion electrospray MS/MS on a Sciex API 4000. The standard curve, which ranged from 5 to 5000 ng/mL, was fitted to a 1/x(2) weighted quadratic regression model for both the BMS drug candidate and its acyl glucuronide. Whole blood and plasma stability experiments were conducted to establish the sample collection, storage, and processing conditions. The validation results demonstrated that this method was rugged and repeatable. The same methodology has also been used in mouse and human plasma for the determination of the BMS drug candidate and its acyl glucuronide.