SMART Digest™ compared with pellet digestion for analysis of human immunoglobulin G1 in rat serum by liquid chromatography tandem mass spectrometry

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

Quantitative analysis of maytansinoid (DM1) in human serum by on-line solid phase extraction coupled with tandem liquid chromatography mass spectrometry - Method validation and its application to clinical samples

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 C18 cartridge. A C18 (150 x 4.6 mm ID 3 µm 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

Fast simultaneous quantitative analysis of FTY720 and its metabolite FTY720-P in human blood by on-line solid phase extraction coupled with tandem liquid chromatography mass spectrometry

Fingolimod (Gilenya®; FTY720), has been recently approved for the treatment of multiple sclerosis in Europe and in the USA. In vivo, FTY720 is phosphorylated by sphingosine kinase 2 to FTY720-phosphate (FTY720-P) which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. The quantitative analysis of FTY720 and that of FTY720-P in blood represents a great challenge. Two separate methods are commonly performed because of the different chemical properties of these two compounds and due the fact that low concentrations have to be measured. In the present study, we have developed and validated a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify FTY720 and FTY720-P in human blood. The sample preparation involves the sample dilution with a solution made of dimethylhexylamine (DMHA), ortho-phosphoric acid and methanol prior to the on-line solid phase extraction (SPE) on a C18 cartridge. The samples were then eluted on a C18 column with a gradient elution of DMHA solution and acetonitrile and analyzed by LC-MS/MS using electrospay ionization in positive mode. The analysis time between 2 samples was 7.5 minutes. Standard curves were linear over the ranges of 0.0800 ng/mL (LLOQ) to 16.0 ng/mL for FTY720 and 0.100 ng/mL (LLOQ) to 20.0 ng/mL for FTY720-P. The method was validated for selectivity, dilution and the accuracy and precision data were in accordance with those specified by the FDA guidance. In addition, stability data obtained during freeze-thaw (3 cycles), at room temperature (24 h), and in an auto-sampler were determined and reported. The method robustness was demonstrated by the consistent data obtained by reanalyzing human blood samples for several clinical studies. In addition comparative data for FTY720 and FTY720-P were obtained between our current method and those of two available separate LC-MS/MS assays. The results of the present work demonstrated that our bioanalytical LC-MS/MS method is rapid, sensitive, specific and reliable for the simultaneous quantitative analysis of FTY720 and FTY720-P in human blood

A fast and reliable reversed phase high performance liquid chromatography method for simultaneous determination of selected anti-retroviral and lumefantrine in human plasma

A fast and reliable high performance liquid chromatography (HPLC) method with UV diode array detection for the simultaneous quantitative analysis of the anti-retroviral drugs, nevirapine (NVP) and efavirenz (EFV) and the anti-malarial, lumefantrine (LUM) in human plasma has been developed and validated. The sample preparation consisted of a plasma protein precipitation with 0.5% acetic acid acetonitrile solution containing the internal standard halofantrine (HALO) prior the LC-analysis. Chromatographic separation was carried out on a Acclaim Polar® Advantage C16, column (150 x 4.6 mm, particle size, 3 µm) using a gradient of mobile phase made of 0.01% TFA in 0.1 M ammonium acetate (solvent A) and 0.1% TFA in acetonitrile (solvent B). The separation of NVP, EFV, LUM and HALO was achieved within 17 min at a flow rate of 1.0 ml min−1 and detections were performed at three wavelengths, 275 nm (NVP), 255 nm (EFV), and 300 nm (LUM). The selectivity of the method was demonstrated in 6 different human plasma batches. In addition, six potential concomitant drugs (zidovudine, pyrimethamine, trimethoprim lamivudine, sulfamethoxazole and fluconazole) were analyzed under our experimental conditions and none of them co-elute with EFV, NVP and LUM. Calibration graphs plotted with seven concentrations in duplicate of each compound were linear between the selected ranges with a regression coefficient (R2) greater than 0.998. Absolute extraction recovery for NVP, EFV and LUM were 99%, 98.6 and 102%, respectively. Inter- and intraday coefficients of variation for LUM, EFV and NVP were ≤10%. The lower limit of quantification was 0.125 μg/mL for LUM and 0.250 μg/mL for both EFV and NVP. Intra- and inter-assay relative standard deviation values were found to be less than 15% at the concentrations examined (0.125–10.0 μg/mL for LUM and 0.250 – 15.0 μg/mL for both EFV and NVP). This low cost method can be used for the simultaneous quantitative analysis of NVP, EFV and LUM concentrations within a clinically relevant concentration range using standard chromatography equipments. This makes our method particularly applicable and useful to resource-limited settings

Laser Diode Thermal Desorption-positive mode Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry for the ultra-fast quantification of a pharmaceutical compound in human plasma

An ultra-fast, reliable and sensitive analytical method enabling high-throughput quantitative analysis of pharmaceutical compounds in human plasma is described. The quantitative work was performed on one of our compound currently under clinical trial by employing a deuterated internal standard (IS). Plasma samples were treated on solid phase micro-extraction (SPME) plates prior their analysis by laser diode thermal desorption and atmospheric pressure chemical ionization coupled to tandem mass spectrometry (LDTD/APCI-MS/MS) in positive mode. The sample analysis run time was 10 seconds as compared to the 7 minutes obtained for the validated LC-MS/MS method. The limit of quantification (LOQ) of the method was estimated at 1 ng/mL. The calibration graphs were linear with a regression coefficient R2 > 0.997. The data of the partial validation show that LDTD/APCI-MS/MS assay was highly reproducible and selective. In addition, the deviations for intra and inter assay accuracy and precision data were within 15% at all quality control levels. The LDTD/APCI-MS/MS method was successfully applied to the analysis of clinical samples and the data obtained were consistent with those found with a validated LC-MS/MS assay. This work demonstrates that LDTD/APCI-MS/MS could be used for the ultra-fast and reliable quantitative analysis of pharmaceutical compounds in human plasma without using the separation step commonly associated with the LC-MS/MS assay

Generic Hybrid Ligand Binding Assay Liquid Chromatography High-Resolution Mass Spectrometry-Based Workflow for Multiplexed Human Immunoglobulin G1 Quantification at the Intact Protein Level: Application to Preclinical Pharmacokinetic Studies

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 <i>in vitro</i> and <i>in vivo</i> 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

Quantitative analysis of hIgG1 in monkey serum by LC-MS/MS using mass spectrometric immunoassay

Background: A sensitive LC-MS/MS method for human immunoglobulin G1 (hIgG1) quantification in monkey serum using mass spectrometric immunoassay disposable automation research tips (MSIA-D.A.R.T.s) is reported. Results: The hIgG1 was captured with biotinylated mouse anti-hIGg1 antibody targeting the fragment crystallizable (Fc) region (50.0 µg/ml) using 1000 cycles. 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 10.0-1000 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. Conclusions: The present assay is suitable for quantitative analysis of hIgG1 based therapeutic (fusion-) proteins in monkey serum at low levels

Ultra-fast quantitative mass spectrometry based method for ceritinib analysis in human plasma and its application for clinical use

Background: We report an ultra-fast method for the quantitative analysis in human plasma of ceritinib, a drug recently approved in the USA for the treatment of non-small-cell lung cancer (NSCLC). Results: Plasma samples were precipitated with acetonitrile containing [13C6]-ceritinib as internal standard and analyzed in 10 seconds each using Laser Diode Thermal Desorption-positive mode Atmospheric Chemical Pressure Ionization tandem mass spectrometry (LDTD-APCI-MS/MS). The method was selective and found to be linear from 5.00-1000 ng/ml using 100 µL plasma. The intra- and inter-days precision and accuracy data met acceptance criteria. The normalized recovery was 69%, carry over and matrix effects were not observed. The method was successfully used to measure clinical samples and consistent data were obtained with the reference LC-ESI-MS/MS method. Conclusions: The developed ultra-fast assay is suitable for quantitative analysis of ceritinib in plasma from clinical trials