Development, validation and application of a liquid chromatography-tandem mass spectrometry method for the activity and inhibition of DPP-4.

Background: Inhibition of the enzyme dipeptidyl peptidase 4 (DPP-4) is a pharmaceutical treatment for type 2 diabetes. To demonstrate bioequivalence of enzyme inhibition of a new dosage form of the inhibitor vildagliptin, a method for enzyme activity was developed, validated and applied using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Results: The method was validated fit for purpose, including accuracy, precision as well as the stability of the activity and the inhibition of DPP-4 in human plasma. Conclusion: A method for the determination of the activity and inhibition of DPP-4 was developed using LC-MS/MS readout; the characteristics and performance of the method met predefined acceptance criteria and were fit for the purpose of a bioequivalence clinical trial.

Monitoring of the deuterated and nondeuterated forms of levodopa and five metabolites in plasma and urine by LC-MS/MS.

To compare pharmacokinetics, metabolism and excretion of levodopa and a triply deuterated form, which is being developed as an improved treatment for Parkinson's disease, methods were needed for quantification of the deuterated and nondeuterated forms of levodopa and five metabolites in human plasma and urine. Results: The natural heavy isotopes in the nondeuterated compounds caused an absolute contribution of up to 100% in the response of the deuterated compounds. Similarly, heavy isotopes in the deuterated analytes contributed to the response of the internal standards, but this did not affect the reliability of the results. Conclusion: Deuterated and nondeuterated analytes can be quantified together by LC-MS/MS, but overestimation of the concentrations of the deuterated molecules may be unavoidable and a careful interpretation of the concentration data is essential.

A quantitative LC-MS/MS method for insulin-like growth factor 1 in human plasma.

BACKGROUND: Insulin-like growth factor 1 (IGF1) is a biomarker with various applications in medicine and also in doping control. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed that employs 15N-IGF1 as an internal standard. The method features urea-based IGF1/IGFBP-complex dissociation which is directly followed by tryptic digestion. Following solid-phase extraction (SPE) sample clean-up of the digest, IGF1 is detected by means of two signature peptides that enable quantification of total IGF1 as well as discrimination between IGF1 proteoforms with 'native' and modified or extended N-terminal sequences. RESULTS: Our method is capable of measuring plasma IGF1 concentrations over the clinically relevant range of 10-1000 ng/mL and was validated according to regulatory guidelines. Comparison with the IDS-iSYS IGF1 immunoassay revealed good correlation (R2>0.97) and no proportional bias between both assays was observed after normalizing the results against the WHO reference standard for IGF1 (02/254). Evaluation of several commercially available IGF1 preparations showed varying responses which were due to inconsistencies in purity and absolute amount of IGF1 present in these products. CONCLUSIONS: Our LC-MS/MS method introduces urea-based dissociation of IGF1/IGFBP-complexes to enable reliable quantification of IGF1 in plasma. Furthermore, the method is able to detect clinically relevant IGF1 levels without an enrichment procedure at the protein-level and thereby minimizes the risk of losing IGF1 proteoforms during sample preparation.

High Sustained Antibody Titers in Patients with Classic Infantile Pompe Disease Following Immunomodulation at Start of Enzyme Replacement Therapy.

OBJECTIVE: To evaluate whether immunomodulation at start of enzyme replacement therapy induces immune tolerance to recombinant human acid alpha-glucosidase (rhGAA) in patients with classic infantile Pompe disease. STUDY DESIGN: Three patients (1 cross reactive immunologic material negative, 2 cross reactive immunologic material positive) were treated with 4 weekly doses of rituximab, weekly methotrexate, and monthly intravenous immunoglobulin and enzyme replacement therapy at 40 mg/kg/week. Antibody titers were measured using enzyme-linked immunosorbent assay. Neutralizing effects on rhGAA activity and cellular uptake were determined and combined with pharmacokinetic analysis. Clinical efficacy was evaluated by (ventilator-free) survival, reduction in left ventricular mass index, and improvement of motor function. RESULTS: Immunomodulation induced B cell depletion that was accompanied by absence of antibody formation in all 3 patients. Upon cessation of rituximab treatment, all 3 patients showed B cell recovery, which was accompanied by formation of very high sustained antibody titers in 2 patients. Neutralizing effects on infused rhGAA were low to mild/moderate. All patients were alive at study end, learned to walk, and showed (near) normalization of left ventricular mass index. CONCLUSIONS: Immunomodulation as recommended in the literature prevented formation of rhGAA antibodies only during B cell depletion but failed to induce immune tolerance in 2 out of 3 patients.

Sensitivity improvement of the LC-MS/MS quantification of carbidopa in human plasma and urine by derivatization with 2,4-pentanedione.

The reliable quantification of carbidopa in biological samples at low concentrations is challenging because of the polar and highly unstable nature of the compound. In this paper, LC-MS/MS methods are described for the determination of carbidopa in 50µL of human plasma and 25µL of human urine in the concentration ranges 1-1,000ng/mL and 100-50,000ng/mL, respectively. After a simple protein precipitation (plasma) or dilution (urine) step, carbidopa is derivatized at its hydrazine moiety by reaction for one hour with 2,4-pentanedione under acidic conditions and at 40°C. The product is a relatively non-polar molecule that is suitable for reversed-phase liquid chromatography (3.5min run time) with detection by tandem mass spectrometry with electrospray ionization. A stable-isotope labeled internal standard is used for response normalization. Precision, accuracy and selectivity of the methods meet the criteria of international guidelines for bioanalytical method validation. Acidification of urine to pH 1.5 and the addition of two anti-oxidants (5mg/mL sodium metabisulfite and 1mg/mL butylated hydroxytoluene) to plasma, in combination with sampling and analysis on ice and under yellow light, ensure sufficient stability of carbidopa. The methods were successfully used to determine plasma pharmacokinetics and urinary excretion of carbidopa in healthy volunteers after a single 37.5mg oral dose.

Absolute quantification of the total and antidrug antibody-bound concentrations of recombinant human α-glucosidase in human plasma using protein G extraction and LC-MS/MS.

The administration of protein-based pharmaceuticals can cause the in vivo formation of antidrug antibodies (ADAs), which may reduce the efficacy of the therapy by binding to the protein drug. An accurate determination of the total and ADA-bound concentrations of the drug gives information on the extent of this immune response and its consequences and may help develop improved therapeutic regimens. We present an absolute quantitative method to differentiate between total, free, and ADA-bound drug for recombinant human alpha acid glucosidase (rhGAA) in plasma from patients suffering from Pompe's disease. LC-MS/MS quantification of a signature peptide after trypsin digestion of plasma samples before and after an extraction of the total IgG content of plasma with protein G coated beads was used to determine the total and the ADA-bound fractions of rhGAA in samples from Pompe patients after enzyme infusion. The methods for total and ADA-bound rhGAA allow quantitation of the drug in the range of 0.5 to 500 µg/mL using 20 µL of plasma and met the regular bioanalytical validation requirements, both in the absence and presence of high levels of anti-rhGAA antibodies. This demonstrates that the ADA-bound rhGAA fraction can be accurately and precisely determined and is not influenced by sample dilution, repeated freezing and thawing, or extended benchtop or frozen storage. In samples from a patient with a reduced response to therapy due to ADAs, high ADA-bound concentrations of rhGAA were found, while in the samples from a patient lacking ADAs, no significant ADA-bound concentrations were found. Since protein G captures the complete IgG content of plasma, including all antidrug antibodies, the described extraction approach is universally applicable for the quantification of ADA-bound concentrations of all non-IgG-based biopharmaceuticals.

High-sensitivity quantitation of a Nanobody® in plasma by single-cartridge multidimensional SPE and ultra-performance LC-MS/MS.

BACKGROUND: A major challenge in protein quantitation based on enzymatic digestion of complex biological samples and subsequent LC-MS/MS analysis of a signature peptide is dealing with the high complexity of the matrix after digestion, which can reduce sensitivity considerably. RESULTS: Using single cartridge multidimensional SPE, sufficient selectivity was introduced to allow quantitation in 50 µl of plasma down to 10.0 ng/ml (~0.3 nM). An inhouse prepared (18)O-labeled signature peptide was used as the internal standard. The procedure was validated for human and rabbit plasma. CONCLUSION: The developed SPE procedure allowed the sensitive and selective LC-MS/MS quantitation of the Nanobody(®) without the use of antibodies. When appropriate precautions are taken, the (18)O-labeled peptide is a practical and economical alternative to custom synthesis.

High-sensitivity LC-MS/MS quantification of peptides and proteins in complex biological samples: the impact of enzymatic digestion and internal standard selection on method performance.

Two important aspects of peptide and protein quantification by LC-MS/MS, the enzymatic digestion step and the internal standardization approach, were systematically investigated with a small protein, salmon calcitonin, which could be analyzed both without and with digestion. Quantification of undigested salmon calcitonin, after solid-phase extraction from plasma, resulted in a lower limit of quantification of 10 pg/mL, while introduction of a tryptic digestion step, followed by quantification of a signature peptide, increased this to 50 pg/mL. The sensitivity was reduced by interferences in the selected reaction monitoring (SRM) transition of the signature peptide due to the increase in sample complexity caused by the digestion and a less selective SRM transition of the signature peptide as compared to undigested salmon calcitonin. Eight internal standardization approaches were compared with respect to accuracy and precision in workflows with and without digestion. Analogue and stable-isotope-labeled (SIL) internal standards were evaluated including an in-house created (18)O-labeled peptide, a cleavable SIL peptide, and an internal standard created by differential derivatization of the signature peptide. We conclude that the best internal standard for the workflows both with and without digestion was the SIL form of the analyte, although the use of several SIL signature peptides and a differentially derivatized signature peptide also resulted in methods with performances which meet the FDA guidelines.

Internal standards in the quantitative determination of protein biopharmaceuticals using liquid chromatography coupled to mass spectrometry.

Following the increase in development of protein biopharmaceuticals, there is a growing demand for the sensitive and reliable quantification of these proteins in complex biological matrices such as plasma and serum to support (pre)-clinical research. In this field, ligand binding assays (LBAs) are currently the standard analytical technique, but in recent years, there is a trend towards the use of liquid chromatography hyphenated with (tandem) mass spectrometry (LC-MS/MS). One of the reasons for this trend is the possibility to use internal standards to correct for analytical variability and thus improve the precision and accuracy of the results. In the LC-MS/MS bioanalysis of small molecules, internal standardization is quite straightforward: either a stable-isotope labeled (SIL) form of the analyte or a structural analogue is used. For the quantification of biopharmaceutical proteins, the situation is more complex. Since the protein of interest is digested to a mixture of peptides, one of which is subsequently used for quantification, there are more options for internal standardization. A SIL form or a structural analogue of either the intact protein or the signature peptide can be used. In addition, a modified form of the SIL-peptide internal standard, containing one or more cleavable groups is a possibility, and an internal standard can be generated during the analysis by using differential derivatization techniques. In this paper we provide an overview of the different options for internal standardization in the field of absolute targeted quantification of protein biopharmaceuticals using LC-MS/MS, based on literature from 2003 to 2011. The advantages and disadvantages of the different approaches are evaluated both with regard to the correction they provide for the variability of the different steps of the analysis and with regard to their generic availability. As most of the approaches used lead to acceptable results in terms of accuracy and precision, we conclude that there currently is no clear preferable method for internal standardization in the field of protein quantification by LC-MS/MS. It is essential, however, that any step in the analysis that is not covered by the internal standard chosen, should be carefully optimized and controlled.

A validated liquid chromatography-tandem mass spectrometry method for the quantitative determination of 4ß-hydroxycholesterol in human plasma.

A novel liquid chromatography-tandem mass spectrometry method is described for the quantitative determination of the endogenous CYP 3A4/5 marker 4ß-hydroxycholesterol in human K(2)-EDTA plasma. It is based on alkaline hydrolysis to convert esterified to free 4ß-hydroxycholesterol, followed by analyte extraction from plasma by hexane and purification of the hexane extract by normal-phase solid-phase extraction. The analyte is chromatographically separated from endogenous isobaric plasma oxysterols and excess cholesterol by a 16-min reversed-phase gradient on a C18 column; detection is performed by atmospheric pressure photoionization tandem mass spectrometry in the positive ion mode, using toluene as a dopant. Using 400µl of plasma, 4ß-hydroxycholesterol can be quantified in the concentration range 10.0-250nM. Validation results show that the method is sufficiently selective towards endogenous plasma sterols and capable of quantifying the analyte with good precision and accuracy. The analyte is sufficiently stable in all relevant matrices and solvents; the addition of the anti-oxidant butylated hydroxytoluene to prevent in vitro formation of 4ß-hydroxycholesterol from cholesterol during storage or analysis is not necessary, provided that long-term frozen storage of plasma occurs at -70°C.