We report the development and the validation of a sensitive liquid chromatography-mass spectrometry (LC-MS/MS) method for mometasone furoate (MF) analysis in human plasma. Plasma samples were processed through liquid-liquid extraction and analyzed using LC-MS/MS operating in positive mode using multiple reaction monitoring of transitions m/z 520.9 → 355.0 and m/z 525.8 → 355.0 for MF and the internal standard (IS), respectively. Separation was achieved at 1.0 mL/min on a C18 column using a gradient elution of mobile phase of 0.05% ammonia in water (phase A) and acetonitrile (phase B). The assay range was 0.250-100 pg/mL and proved to be accurate and precise MF. Normalized recoveries were consistent and reproducible with a coefficient of variation (CV%) value of 6.0. The CV (%) of the IS normalized matrix factor was not observed in normal, lipemic, and hemolyzed plasmas. Dilutions of 1:10 were accurately quantified. A cycle of three freeze and thaw and stabilities at room temperature and on the autosampler were demonstrated. In addition, MF in the presence of indacaterol and glycopyrronium was proven to be stable at -70°C for at least 157 days. The present method was successfully applied to quantify MF in patients receiving MF, indacaterol, and glycopyrronium as a fixed-dose combination.

Sample preparation is essential for low-level compound determination. In the present work, supported liquid extraction (SLE) was used as sample preparation for the low-level determination of a new TLR7 agonist imiquimod compound, LFX453. Samples were extracted on ISOLUTE® SLE 96-well plates using tert-butyl-methyl ether followed by evaporation and dry residue reconstitution with 150 μl of a mixture of 0.1% formic acid in acetonitrile-water (50/50, v/v). Samples were eluted using a flow rate of 0.750 ml/min on a C18 column (50 × 2.1 mm, 2.7 μm) with a mobile phase consisting of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Tandem mass spectrometry was used to analyze the samples in positive mode. The method run time was 6.5 min, and the low limit of quantification was 1.00 pg/ml with 0.100 ml of minipig plasma. Intra-run and inter-run precision and accuracy were within the acceptance criteria at four concentration levels over a concentration ranging from 1.00 to 200 pg/ml. There was no matrix effect and recovery, three freeze-thaw cycles and incurred samples reanalysis were validated. The method was successfully applied for measuring LFX453 in minipig plasma after application on minipig skin.

Background A once-daily (o.d.) fixed-dose combination of indacaterol acetate (IND), glycopyrronium bromide (GLY), and mometasone furoate (MF) delivered via the Breezhaler ® device (IND/GLY/MF) is being developed for treatment of asthma. This study compared steady-state pharmacokinetics of IND, GLY and MF between Japanese and Caucasian male subjects after multiple inhalations of IND/GLY/MF o.d. Methods This was a single-center, open-label, 2-treatment crossover study with a 21-day washout period. Japanese and Caucasian subjects received IND/GLY/MF 150/50/80 μg (inhaled corticosteroid [ICS] medium-dose) or 150/50/160 μg o.d. (ICS high-dose) for 14 days in each period. Pharmacokinetics were characterized up to 24 h post-dose on Days 1 and 14. Results In total, 16 Japanese (median age 31 years [range 20–40 years], mean weight 68.3 kg) and 17 Caucasian subjects (median age 27 years [range 21–43 years], mean weight 75.0 kg) were randomized. Geometric mean ratios (Japanese/Caucasian) [90% confidence interval (CI)] for C max for IND, GLY and MF at the high ICS dose on Day 14 were 1.31 [1.13, 1.51] 1.38 [1.13, 1.69] and 1.07 [0.969, 1.18], respectively. Geometric mean ratios (Japanese/Caucasian) [90% CI] for AUC 0–24h on Day 14 for IND, GLY and MF at the high ICS dose were 1.17 [1.01, 1.35], 1.05 [0.920, 1.20] and 1.15 [1.05, 1.27] respectively. Similar trends were noted for all components for the medium ICS dose treatment. IND/GLY/MF was safe and well tolerated; no AEs suspected to be study drug-related were observed. Conclusion Pharmacokinetics of IND, GLY and MF (high and medium dose) when delivered as a fixed-dose combination were comparable between Japanese and Caucasian subjects. The IND/GLY/MF combination at the administrated doses was safe and well tolerated in both ethnic groups. Trial registration Japan Registry of Clinical Trial: jRCT2031200227, retrospectively registered on 04, December, 2020.

BioanalysisVol. 12, No. 2 EditorialFree AccessMatrix matching in quantitative bioanalysis by LC–MS/MS a dream or a reality?Olivier Heudi, Serge Winter & Franck PicardOlivier Heudi*Author for correspondence: Tel.: +41 79 53 59 611; E-mail Address: Olivier.heudi@novartis.comNovartis Pharma AG, DMPK/Bioanalytics, CH-4056 Basel, Switzerland, Serge WinterNovartis Pharma AG, DMPK/Bioanalytics, CH-4056 Basel, Switzerland & Franck PicardNovartis Pharma AG, DMPK/Bioanalytics, CH-4056 Basel, SwitzerlandPublished Online:19 Dec 2019https://doi.org/10.4155/bio-2019-0273AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Keywords: internal standardLC–MSLC–MS/MS quantitationmatrix effectvalidationLC–MS/MS quantitative analysis & ESILiquid chromatography coupled with atmospheric pressure ionization tandem MS (LC–MS/MS) is widely used for the quantitative analysis of small molecules in pharmaceutical industry. The high sensitivity and selectivity of LC–MS/MS apparatus have enabled the development of methods with minimum sample preparation, short separation time and detection in ESI mode in an environment where a large amount of endogenous compounds can co-exist with the analyte of interest [1–3]. However, it has been clearly shown that when ESI is used, the intensity of the protonated molecule of organic base decreases significantly in the presence of an increasing amount of another basic compound [1,4]. These co-elute compounds could significantly affect the analyte MS response and the accuracy of the quantitative data.In sample internal standard monitoring & in sample calibration curveIn the US FDA Bioanalytical Method Validation Guidance for Industry [5] and the EMA Guideline on Bioanalytical Method Validation [6], it is stated that calibration standards have to be prepared in the same biological matrix as the samples in the intended study. Moreover, matrix effect and recovery has to be determined in hemolyzed, hyperlipidemic plasma samples in case anticipated in the study [6]. In addition, investigations has to be performed in plasma sample from special populations (renally or hepatically impaired). Another point to consider is the impact of formulation excipients on concentration data obtained on the early time point for samples collected after infusion or of intravenous administration [6]. However, not all these approaches are without their limitations. Most spiked samples fail to mimic the incurred samples as each sample has co-eluting, interfering compounds that are thereby exposed to a different extent of ionization suppression [7]. Ultimately, each study sample should be treated as a unique one. Therefore, is it practical to rely on an exact matrix matching in calibration standards with the samples, when each sample contains different co-eluting compounds at different concentrations and time points? One way to correct for the analyte MS signal suppression or enhancement is to use during the sample analysis an internal standard (IS) – preferably a (stable isotope labeled SIL) analog, which has identical chemical and physical properties to the target analyte [8]. Since it is processed along with the analyte, this IS should compensate for the losses that may occur during the sample preparation. In a recent guidance, the FDA recommends to check the IS responses in each analyzed samples and to further investigate on samples with the IS signals that deviate from the rest of the spiked samples prepared in the same matrix [9]. However, performing these investigations remain a daunting challenge as one can only speculate on the origin of the IS signal variability due the complexity of each plasma sample composition. Hence, it would be unfair to discard sample PK concentrations based on abnormal IS response. In reality, this approach is not in line with attributed function of the IS in the quantitative analysis by LC–MS/MS. Since the IS has the same physical properties to that of the analyte and the IS co-elute, both will experience the same sort of ionization (enhancement or suppression); hence, the analyte to IS peak area (height) ratio remains unaffected [10]. In a recent study where IgG were determined in preclinical species by LC–MS/MS using the bottom-up approach, we have demonstrated that accurate data were obtained when measuring quality control (QC) spiked monkey plasma samples against a calibration curve prepared in rat plasma, despite the IS signal difference observed with the two plasma matrices [11]. This will mean that there is no need to adopt the matrix matching concept and that a generic matrix can be used to generate external calibration curves. Consequently, a unique matrix, human plasma for example, can be considered to analyze samples from other species such as monkey, rat or dog, providing that a proper IS, preferably labeled with 13C or 15N atoms with identical physical properties (same elution time and ionization pattern) to that of the analyte is used. This approach will avoid the use of multiple matrices and preclude the development of surrogate matrices when handling rare matrices.In realizing the impracticality of exact matrix matching, Gu et al. [12] recently reported a novel in-sample calibration curve (ISCC) methodology. In this approach, multiple naturally occurring isotopologs of stable isotopically labeled IS spiked in the sample at known concentration is used to build the calibration curve in each sample. Hence, there is no need to use external calibration obtained in a similar matrix and this eliminates the difference between the calibration standard and unknown sample matrices. ISCC will considerably simplify the workout procedure for quantitative analysis by LC–MS/MS, especially for study containing high number of samples and originating from various sources (healthy volunteer, gender, etc.) where assessing the matrix is just not possible.¨ConclusionThe concept one-matrix-fits-all samples (one external calibration curve to measure all incurred sample – with the exception of special cases) led the analyst to carry several matrices and to perform tedious assessments such as matrix effect or to develop surrogate matrices. However, these assessments will not always help understanding the behavior of the analyte in incurred sample matrices. Since we are moving toward the concept of 'personalized incurred samples', it is important to monitor the IS signal during the sample analysis, and if abnormal signal is observed, assess each case without necessarily rejecting the sample concentration found. In fact, in the unknown samples displaying abnormal IS signal, the MS ionization will affect in a similar manner the analyte of interest and its corresponding IS. The advancements made in analytical technology and the development of novel approach such ISCC will help revisiting, simplifying and changing guidelines that will enable analysts to take full advantage of the new developments to cut down the time and cost associated with routine analysis.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.References1. Kebarle P, Tang L. From ions in solution to ions in the gas-phase – the mechanism of electrospray mass-spectrometry. Anal. Chem. 65(22), A972–A986 (1993).CAS, Google Scholar2. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Matrix effect in quantitative LC/MS/MS analyses of biological fluids: a method for determination of finasteride in human plasma at picogram per milliliter concentrations. Anal. Chem. 70(5), 882–889 (1998).Crossref, Medline, CAS, Google Scholar3. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal. Chem. 75(13), 3019–3030 (2003).Crossref, Medline, CAS, Google Scholar4. Gosetti F, Chiuminatto U, Zampieri D, Mazzucco E, Marengo E, Gennaro MC. A new on-line solid phase extraction high performance liquid chromatography tandem mass spectrometry method to study the sun light photodegradation of mono-chloroanilines in river water. J. Chromatogr. A. 1217(20), 3427–3434 (2010).Crossref, Medline, CAS, Google Scholar5. US FDA. Bioanalytical method validation guidance for industry (2018). www.Fda.Gov/Downloads/Drugs/Guidancecomplianceregulatoryinformation/Guidances/Ucm070107.PdfGoogle Scholar6. EMA. Guideline on bioanalytical method validation (2011). www.Ema.Europa.Eu/Docs/En_Gb/Document_Library/Scientific_Guideline/2011/08/Wc500109686.PdfGoogle Scholar7. Hewavitharana AK. Matrix matching in liquid chromatography–mass spectrometry with stable isotope labelled internal standards – is it necessary? J. Chromatogr. A. 1218(2), 359–361 (2011).Crossref, Medline, CAS, Google Scholar8. Van Eeckhaut A, Lanckmans K, Sarre S, Smolders I, Michotte Y. Validation of bioanalytical LC–MS/MS assays: evaluation of matrix effects. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 877(23), 2198–2207 (2009).Crossref, Medline, CAS, Google Scholar9. US FDA. Evaluation of internal standard responses during chromatographic bioanalysis: questions and answers guidance for industry (2019). www.Fda.Gov/Drugs/Guidancecomplianceregulatoryinformation/Guidances/Default.HtmGoogle Scholar10. Liang HR, Foltz RL, Meng M, Bennett P. Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17(24), 2815–2821 (2003).Crossref, Medline, CAS, Google Scholar11. Lanshoeft C, Wolf T, Walles M et al. The flexibility of a generic LC–MS/MS method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G and related constructs in animal studies. J. Pharm. Biomed. Anal. 131, 214–222 (2016).Crossref, Medline, CAS, Google Scholar12. Gu HD, Zhao Y, Demichele M et al. In-sample calibration curve using multiple isotopologue reaction monitoring of a stable isotopically labeled analyte for instant LC–MS/MS bioanalysis and quantitative proteomics. Anal. Chem. 91(3), 2536–2543 (2019).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByBinding the gap between experiments, statistics, and method comparison: A tutorial for computing limits of detection and quantification in univariate calibration for complex samplesAnalytica Chimica Acta, Vol. 1209Welcome to volume 13 of BioanalysisSankeetha Nadarajah20 January 2021 | Bioanalysis, Vol. 13, No. 1 Vol. 12, No. 2 Follow us on social media for the latest updates Metrics Downloaded 2,025 times History Received 18 October 2019 Accepted 7 November 2019 Published online 19 December 2019 Published in print January 2020 Information© 2020 Newlands PressKeywordsinternal standardLC–MSLC–MS/MS quantitationmatrix effectvalidationFinancial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download

We report a selective LC-MS/MS method for the simultaneous quantitative determinations of the adenosine A2a receptor antagonist NIR178 (NIR178) and its major metabolite NJI765 in human plasma. Sample preparation steps involved protein precipitation, sample evaporation and reconstitution using a plasma sample volume of 0.1 ml plasma. Separation was achieved in 10 min on an Acquity UPLC BEH C18 1.7 μm, 2.1 × 50 mm column heated at 60°C with a gradient elution at 0.6 ml/min mobile phase made of water and acetonitrile both acidified with 0.1% formic acid. The detection was performed in positive ion mode and quantification based on multiple reaction monitoring. The linear response range was 1.00-1,000 ng/ml using a 1/x2 weighting factor. The intra- and inter-day accuracies (bias %) and intra- and inter-day precisions (CV, %) obtained for NIR178 and NJI765 were within the acceptance criteria. The normalized NIR178 and NJI765 matrix factor calculated from six lots of normal, lipemic and hemolyzed plasmas ranged from 0.97 to 1.05. The normalized recoveries of both NIR178 and NJI765 compared with their internal standards were consistent and reproducible with a CV ≤8.0. This method was successfully applied to support pharmacokinetic studies in adult patients with cancer.

Indacaterol (IND), is co-formulated with glycopyrronium (GLY), and mometasone furoate (MF) as a once-daily (o.d.) inhaled fixed-dose combination (IND/GLY/MF) delivered via the Breezhaler (R) device for maintenance treatment of asthma. We evaluated the steady state plasma pharmacokinetics (PK) of IND, GLY and MF following inhalation of IND/GLY/MF or as monotherapies. This was a randomized, open-label, four-way crossover study. Subjects received IND/GLY/MF 150/50/160 mu g (high-dose), IND 150 mu g, GLY 50 mu g or MF 190 mu g (in vitro fine particle mass comparable to 160 mu g MF in IND/GLY/MF) via the Breezhaler (R) device, o.d. for 14 days in each period, with a washout of at least 7 days. PK was characterized on Day 14, up to 24 h post-dose. In total, 36 healthy subjects were randomized. For IND, the geometric mean ratios (90% CI) for AUC0-24h,ss and Cmax,ss were 0.922 (0.878, 0.969) and 1.02 (0.967, 1.08), respectively for the IND/GLY/MF versus IND monotherapy comparison. For GLY, the geometric mean ratios (90% CI) for AUC0-24h,ss and Cmax,ss were 0.986 (0.944, 1.03) and 1.21 (1.09, 1.34), respectively for the IND/GLY/MF versus GLY comparison. For MF, the geometric mean ratios (90% CI) for AUC0-24h,ss and Cmax,ss were 1.16 (1.09, 1.24) and 1.17 (1.09, 1.25), respectively for IND/GLY/MF versus MF comparison. Similar systemic exposure was noted for IND/GLY/MF versus monotherapy for all three mono-components, indicating a lack of PK interaction. Multiple inhaled doses of IND, GLY and MF were safe and well tolerated, when administered alone or in combination. There was no clinically relevant pharmacokinetic interaction between IND, GLY and MF when administered as IND/GLY/MF.

Quantitation of recombinant monoclonal antibodies (mAb) using liquid chromatography tandem mass spectrometry LC-MS/MS for pharmacokinetic (PK) studies is becoming a complementary approach to traditional antibody-based ligand binding assays (LBA). Here we describe the production of stable isotope labeled human immunoglobulin G1 (hIgG1) mAb for use as an internal standard (IS) for LC-MS/MS-based antibody quantitation. Stable isotope labeling with amino acids in cell-culture (SILAC) allowed production of [13C6-L-Lysine (Lys),13C6-L-Arginine (Arg)]-mAb. A fermentation process was run in shake flasks containing defined medium, so that the targeted stable isotope labeled amino acids were incorporated into the recombinant monoclonal antibody (mAb). The mAb was then purified and label incorporation was determined to be >99% of all Lys and Arg moieties by mass spectrometry. Sequence coverage was confirmed by peptide mapping. The full length antibody was then used as IS to assist the development of a generic LC-MS/MS method for the quantitative analysis of human IgG1 in rat serum. Four tryptic peptides (GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), FNWYVDGVEVHNAK (FNW) and VVSVLTVLHQDWLNGK (VVS)) originating from the Fragment crystallizable (Fc) region of a selected IgG1 were used for quantitation and they were chromatographically resolved. In addition, the calibration curves resulting from the peak area ratio of each peptide to its respective labeled IS over the range of 1-1000 µg/mL (GPS, VVS and TTP) or 5-1000 µg/mL (FNW) were reproducible and good accuracy precision data were obtained on calibrators (Cs) and quality control samples (QCs). The produced full length stable isotope labeled monoclonal antibody was found to be a suitable IS to support the development of generic method for IgG1 quantitation in rat serum. Our data demonstrated that this antibody, used as a universal IS, can allow for more rapid, accurate and cost effective quantitation of fully human and human IgG1 in preclinical species.

The quantitative analysis of human immunoglobulin G1 (hIgG1) by mass spectrometry is commonly performed using surrogate peptides after enzymatic digestion. Since some limitations are associated with this approach, a novel workflow is presented by hybridizing ligand binding assay (LBA) with liquid chromatography-high-resolution mass spectrometry (LC-HRMS) for hIgG1 quantification directly at the intact protein level. Different hIgG1s, including a [13C]-labeled version used as internal standard, were immuno-enriched from rat serum with a fully automated platform based on streptavidin coated tips and a biotinylated mouse anti-hIgG capture antibody targeting the fragment crystallizable region followed by overnight deglycosylation prior to LC-HRMS analysis. The proposed quantitative workflow utilized extracted ion chromatograms (XICs) from the nondeconvoluted full-scan MS spectrum. The assay was validated in terms of selectivity, sensitivity, accuracy/precision, carry-over, dilution linearity, and reproducibility. Consistent data between the conventional approach based on surrogate peptide analysis and our proposed workflow were obtained in vitro and in vivo with the advantage of a less extensive sample pretreatment. Multiplexing capabilities for simultaneous quantification of different hIgG1s within the same spiked sample were also exemplified. Altogether our results pave the way not only for the thorough application of intact hIgG1 quantification by LBA-LC-HRMS but also as a generic quantitative analytical method for other hIgG isotypes or next generation biotherapeutics.

The newly developed SMART Digest™ kit was applied for the sample preparation of human immunoglobulin G1 (hIgG1) in rat serum prior to qualitative and quantitative analyses by liquid chromatography tandem mass spectrometry (LC-MS/MS). The sequence coverages obtained for the light and heavy chains of hIgG1A were 50 and 76%, respectively. The calibration curve was linear from 1.00 to 1000 μg/ml for three of four generic peptides. Overall, the SMART Digest™ kit resulted in similar quantitative data (linearity, sensitivity, accuracy, and precision) compared with the pellet digestion protocol. However, the SMART Digest™ required only 2 h of sample preparation with fewer reagents.

For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs to be well understood to mitigate toxicity risks and properly design the first in-patient studies. Therefore, a distribution, metabolism, and excretion (DME) study with a radiolabeled rat cross-reactive ADC ([(3)H]DM1-LNL897) targeting the P-cadherin receptor was conducted in female tumor-bearing nude rats. Although multiple components [total radioactivity, conjugated ADC, total ADC, emtansine (DM1) payload, and catabolites] needed to be monitored with different technologies (liquid scintillation counting, liquid chromatography/mass spectrometry, enzyme-linked immunosorbent assay, and size exclusion chromatography), the pharmacokinetic data were nearly superimposable with the various techniques. [(3)H]DM1-LNL897 was cleared with half-lives of 51-62 hours and LNL897-related radioactivity showed a minor extent of tissue distribution. The highest tissue concentrations of [(3)H]DM1-LNL897-related radioactivity were measured in tumor. Complimentary liquid extraction surface analysis coupled to micro-liquid chromatography-tandem mass spectrometry data proved that the lysine (LYS)-4(maleimidylmethyl) cyclohexane-1-carboxylate-DM1 (LYS-MCC-DM1) catabolite was the only detectable component distributed evenly in the tumor and liver tissue. The mass balance was complete with up to 13.8% ± 0.482% of the administered radioactivity remaining in carcass 168 hours postdose. LNL897-derived radioactivity was mainly excreted via feces (84.5% ± 3.12%) and through urine only to a minor extent (4.15% ± 0.462%). In serum, the major part of radioactivity could be attributed to ADC, while small molecule disposition products were the predominant species in excreta. We show that there is a difference in metabolite profiles depending on which derivatization methods for DM1 were applied. Besides previously published results on LYS-MCC-DM1 and MCC-DM1, maysine and a cysteine conjugate of DM1 could be identified in serum and excreta.

In the present study, the application of a liquid chromatography high-resolution mass spectrometry (LC-HRMS) analytical assay for the quantitative analysis of a recombinant human immunoglobulin G1 (hIgG1) in rat serum is reported using three generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), and VVSVLTVLHQDWLNGK (VVS). Moreover, the deamidation site of a fourth peptide FNWYVDGVEVHNAK (FNW) was identified and further excluded from the assay evaluation due to the inaccuracy of the quantitative results. The rat serum samples were spiked with a fully labeled hIgG1 as internal standard (ISTD). The digestion with trypsin was performed onto the pellet prior to peptide analysis by LC-HRMS using a quadrupole time of flight (QTOF) mass analyzer operating in selected reaction monitoring (SRM) mode with enhanced duty cycles (EDC). The assay linearity for the three investigated peptides was established for a hIgG1 (hIgG1A) from 1.00 to 1000 μg mL−1 with a mean coefficient of determination (R 2) higher than 0.9868. The inter-day accuracy and precision obtained in rat serum over 3 days were ≤11.4 and ≤10.5 %, respectively. Short-term stability on the auto-sampler at 6 °C for 30 h, at RT for 48 h, and a 100-fold dilution factor were demonstrated. In addition, QC samples prepared in cynomolgus monkey serum and measured with the present method met the acceptance criteria of ±20.0 and ≤20.0 % for all three peptides regarding accuracy and precision, respectively. The LC-HRMS method was applied to the analysis of samples from five individual cynomolgus monkeys dosed with a second hIgG1 (hIgG1B) and consistent data were obtained compared to the LC-MS/MS method (conventional triple quadrupole (QqQ) mass analyzer operating in SRM). The present data demonstrate that LC-HRMS can be used for the quantitative analysis of hIgG1 in both species and that quantification is not only limited to classical QqQ instruments.

AIM:A sensitive generic LC-MS/MS method for hIgG1 quantification in cynomolgus monkey serum using mass spectrometric immunoassay disposable automation research tips (MSIA-D.A.R.T.'S™) is reported.RESULTS:The hIgG1 was captured with a biotinylated mouse anti-hIgG antibody (50.0 µg/ml) targeting the fragment crystallizable (Fc) region. Elution from the streptavidin-coated MSIA-D.A.R.T.'s was conducted with 0.4% trifluoroacetic acid in water. The method was selective and linear from 10.0 to 1000 ng/ml using 100 µl of serum. The method was evaluated regarding accuracy, precision, carry-over, dilution, auto-sampler stability and applied for the determination of hIgG1 concentration in monkey serum after intravitreal administration.CONCLUSION:The present assay is suitable for quantitative analysis of hIgG1-based therapeutic proteins in monkey serum at low levels.

An increasing demand of new analytical methods is associated with the growing number of biotherapeutic programs being prosecuted in the pharmaceutical industry. Whilst immunoassay has been the standard method for decades, a great interest in assays based on liquid chromatography tandem mass spectrometry (LC-MS/MS) is evolving. In this present work, the development of a generic method for the quantitative analysis of therapeutic proteins based on human immunoglobulin G (hIgG) in rat serum is reported. The method is based on four generic peptides GPSVFPLAPSSK (GPS), TTPPVLDSDGSFFLYSK (TTP), VVSVLTVLHQDWLNGK (VVS) and FNWYVDGVEVHNAK (FNW) originating from different parts of the fraction crystallizable (Fc) region of a reference hIgG1 (hIgG1A). A trsrptic pellet digestion of rat serum spiked with hIgG1A and a stable isotope labeled protein (hIgG1B) used as internal standard (ISTD) was applied prior LC-MS/MS analysis. The upper limit of quantification was at 1000 mu g/mL The lower limit of quantitation was for GPS, TTP and VVS at 1.00 mu g/ml. whereas for FNW at 5.00 mu g/mL. Accuracy and precision data met acceptance over three days. The presented method was further successfully applied to the quantitative analysis of other hlgGls (hIgG1C and hIgG1D) and hIgG4-based therapeutic proteins on spiked quality control (QC) samples in monkey and rat serum using calibration standards (Cs) prepared with hIgG1A in rat serum. In order to extend the applicability of our generic approach, a bispecific-bivalent hlgG1 (bb-hIgG1) and two lysine conjugated antibody-drug conjugates (ADC1 and ADC2) were incorporated as well. The observed values on spiked QC samples in monkey serum were satisfactory with GPS for the determination of bb-hIgG1 whereas the FNW and UP peptides were suitable for the ADCs. Moreover, comparable mean concentration-time profiles were obtained from monkeys previously dosed intravenously with ADC2 measured against Cs samples prepared either with hIgG1A in rat serum (presented approach) or with the actual ADC2 in monkey serum (conventional approach). The results of this study highlight the great flexibility of our newly developed generic approach and that the choice of the surrogate peptide still remains critical when dealing with different matrix types or modalities. (C) 2016 Elsevier B.V. All rights reserved.

Here, we describe the production of stable isotope-labeled human immunoglobulin G1 ([13 C]-hIgG1) using [13 C]-L-lysine/arginine-labeled hIgG1. The fermentation process was run in shake flasks containing labeled arginine and lysinethat were incorporated into the produced recombinant hIgG1. The [13 C]-hIgG1 was purified, and label incorporation was determined to be >99% at all lysine and arginine moieties. Sequence coverage was confirmed by peptide mapping. [13 C]-hIgG1 was then used as an internal standard (IS) for the development of a liquid chromatography-tandem mass spectrometry method applicable to the quantitative analysis of all human types of hIgG1 in rat serum. Four conserved peptides, namely, GPSVFPLAPSSK, TTPPVLDSDGSFFLYSK, VVSVLTVLHQDWLNGK, and FNWYVDGVEVHNAK, originating from different parts of the fraction crystallizable region of hIgG1, were used for quantitation of hIgG1 in rat serum. The calibration curves with a coefficient of determination (r2 ) between 0.9950 and 0.9962 resulting from the peak area ratio of each peptide to its respective labeled IS were reproducible. A mean bias within ±20.0% of the nominal values and a precision of ≤20.0 % were obtained for the calibration standards and quality control samples for each peptide. [13 C]-hIgG1 was shown as a suitable IS for quantitative hIgG1 analysis in preclinical species by LC-MS/MS.

A sensitive and specific method was developed and validated for the quantitation of maytansinoid (DM1) in human serum using on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Because DM1 contains a free thiol moiety, likely to readily dimerize or react with other thiol-containing molecules in serum, samples were pre-treated with a reducing agent [tris (2-carboxyethyl) phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM). The resulting samples were diluted with acetonitrile prior to the on-line solid phase extraction (SPE) on a C-18 cartridge. A C-18 (150 x 4.6 mm ID 3 mu m particle size) column was used for chromatographic separation with a 10.0 min HPLC gradient and DM1-NEM was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. DM1 concentrations were back-calculated from DM1-NEM amount found in the human serum samples. The quantitation range of the method was 0.200-200 ng/mL when using 0.25 mL serum. Within-run day precisions (n = 6) were 0.9-4.4% and between-run day (3 days runs; n = 18) precisions 2.5-5.6%. Method biases were between 3.5-14.5% across the whole calibration range. DM1-NEM exhibited sufficiently stability under all relevant analytical conditions and no DM1 losses from the ADC were observed. Finally, the assay was used for DM1 determination in human serum concentration after the intravenous administration of an investigational antibody drug conjugate (ADC) containing DM1 as payload. (C) 2015 Elsevier B.V. All rights reserved.

Hematocrit (Hct) is one of the most critical issues associated with the bioanalytical methods used for dried blood spot (DBS) sample analysis. Because Hct determines the viscosity of blood, it may affect the spreading of blood onto the filter paper. Hence, accurate quantitative data can only be obtained if the size of the paper filter extracted contains a fixed blood volume. We describe for the first time a microfluidic-based sampling procedure to enable accurate blood volume collection on commercially available DBS cards. The system allows the collection of a controlled volume of blood (e.g., 5 or 10 μL) within several seconds. Reproducibility of the sampling volume was examined in vivo on capillary blood by quantifying caffeine and paraxanthine on 5 different extracted DBS spots at two different time points and in vitro with a test compound, Mavoglurant, on 10 different spots at two Hct levels. Entire spots were extracted. In addition, the accuracy and precision (n = 3) data for the Mavoglurant quantitation in blood with Hct levels between 26% and 62% were evaluated. The interspot precision data were below 9.0%, which was equivalent to that of a manually spotted volume with a pipet. No Hct effect was observed in the quantitative results obtained for Hct levels from 26% to 62%. These data indicate that our microfluidic-based sampling procedure is accurate and precise and that the analysis of Mavoglurant is not affected by the Hct values. This provides a simple procedure for DBS sampling with a fixed volume of capillary blood, which could eliminate the recurrent Hct issue linked to DBS sample analysis.

AIM:An ultrafast, sensitive, selective and robust LDTD-APCI-MS/MS method was developed for the quantification of ceritinib in human plasma. RESULTS:Samples were protein precipitated using acetonitrile containing [(13)C6]-ceritinib as internal standard. The assay was validated over a concentration range from 5.00 to 1000 ng/ml. Intra- and inter-day precision and accuracy met acceptance from EMA and US FDA guidelines. The normalized recovery was 69%, whereas no carryover and matrix effects were observed. The method was applied to clinical samples and resultant data were consistent with the LC-ESI-MS/MS reference method. CONCLUSION:The new assay is suitable for ceritinib quantification in clinical trials, whereas the analysis time is significantly reduced to 10 s.

A sensitive and ultra-fast method utilizing the laser diode thermal desorption ion source using atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD-APCI-MS/MS) was developed for the quantitative analysis of BKM120, an investigational anticancer drug in human plasma. Samples originating from protein precipitation (PP) followed by salting-out assisted liquid-liquid extraction (SALLE) were spotted onto the LazWell™ plate prior to their thermal desorption and detection by tandem mass spectrometry in positive mode. The validated method described in this paper presents a high absolute extraction recovery (>90 %) for BKM120 and its internal standard (ISTD) [D8]BKM120, with precision and accuracy meeting the acceptance criteria. Standard curves were linear over the range of 5.00 to 2000 ng mL−1 with a coefficient of determination (R 2) >0.995. The method specificity was demonstrated in six different batches of human plasma. Intra- and inter-run precision as well as accuracy within ±20 % at the lower limit of quantification (LLOQ) and ±15 % (other levels) were achieved during a three-run validation for quality control (QC) samples. The post-preparative stability on the LazWell™ plate at room temperature was 72 h and a 200-fold dilution of spiked samples was demonstrated. The method was applied successfully to three clinical studies (n = 847) and cross-checked with the validated LC-ESI-MS/MS reference method. The sample analysis run time was 10 s as compared to 4.5 min for the current validated LC-ESI-MS/MS method. The resultant data were in agreement with the results obtained using the validated reference LC-ESI-MS/MS assay and the same pharmacokinetic (PK) parameters were calculated for both analytical assays. This work demonstrates that LDTD-APCI-MS/MS is a reliable method for the ultra-fast quantitative analysis of BKM120 which can be used to speed-up and support its bioanalysis in the frame of the clinical trials.