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Quantitation of leukotriene B(4) in human sputum as a biomarker using UPLC-MS/MS.

Abstract

Leukotriene B4 (LTB4) is a potent mediator of inflammation and has been recognized as an important target for therapeutic intervention for treatment of diseases such as asthma. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of LTB4 in human sputum as a biomarker for LTB4 biosynthesis inhibition. A fit-for-purpose strategy for method development, assay qualification, and study support was adopted for this biomarker project. A surrogate matrix (protein buffer) was used for preparation of calibration samples and certain levels of quality control (QC) samples to avoid interference from endogenous analyte, while the low QC was prepared in authentic matrix, human sputum. The analytical methodology utilized a liquid-liquid extraction procedure in 96-well plate format. Chromatographic separation was achieved with a reversed-phase ultra high pressure liquid chromatography (UPLC) column using gradient elution, and the run time was 4.5min per sample. The lower limit of quantitation (LLOQ) was 0.2ng/mL, and the calibration curve range was 0.2-20ng/mL. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (6h), freeze-thaw stability (3 cycles at -20°C), and autosampler stability (97h at ambient temperature) all met acceptance criteria. Frozen long-term stability for 166 days at -20°C in sputum did not meet acceptance criteria by showing only ≥75% of nominal concentration and the information was taken into consideration for study support. Two important observations in the current work were: (1) LTB4 was unstable in sputum in the presence of liquification reagent dithiothreitol (DTT). Therefore, a non-DTT treatment method for sputum processing was developed and applied to the bioanalytical assay and clinical study support; and (2) chromatographic separation of LTB4 from its three non-enzymatically derived isomers, i.e. 6-trans-LTB4, 12-epi-LTB4, and 6-trans-12-epi-LTB4, was achieved. This assay was successfully applied to a Phase II clinical study for proof-of-concept of a LTA4 hydrolase inhibitor for treatment of asthma.

Authors+Show Affiliations

Drug Safety Sciences, Janssen Research & Development, Johnson & Johnson, Raritan, NJ 08869, USA. wjian@its.jnj.comNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Evaluation Study
Journal Article

Language

eng

PubMed ID

23831697

Citation

Jian, Wenying, et al. "Quantitation of Leukotriene B(4) in Human Sputum as a Biomarker Using UPLC-MS/MS." Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, vol. 932, 2013, pp. 59-65.
Jian W, Edom RW, Xue X, et al. Quantitation of leukotriene B(4) in human sputum as a biomarker using UPLC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2013;932:59-65.
Jian, W., Edom, R. W., Xue, X., Huang, M. Q., Fourie, A., & Weng, N. (2013). Quantitation of leukotriene B(4) in human sputum as a biomarker using UPLC-MS/MS. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences, 932, 59-65. https://doi.org/10.1016/j.jchromb.2013.06.010
Jian W, et al. Quantitation of Leukotriene B(4) in Human Sputum as a Biomarker Using UPLC-MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2013 Aug 1;932:59-65. PubMed PMID: 23831697.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitation of leukotriene B(4) in human sputum as a biomarker using UPLC-MS/MS. AU - Jian,Wenying, AU - Edom,Richard W, AU - Xue,Xiaohua, AU - Huang,Mike-Qingtao, AU - Fourie,Anne, AU - Weng,Naidong, Y1 - 2013/06/20/ PY - 2013/04/01/received PY - 2013/06/03/revised PY - 2013/06/07/accepted PY - 2013/7/9/entrez PY - 2013/7/9/pubmed PY - 2014/2/4/medline KW - BQL KW - Bioanalysis KW - Biomarker KW - COPD KW - DTT KW - Fit-for-purpose KW - HQC KW - HSA KW - LLE KW - LLOQ KW - LQC KW - LT KW - LTA(4) KW - LTB(4) KW - Leukotriene B4 KW - MQC KW - MRM KW - MS KW - MTBE KW - PBS KW - QC KW - Sputum KW - UPLC KW - UPLC–MS/MS KW - below quantitation limit KW - chronic obstructive pulmonary disease KW - dithiothreitol KW - high QC KW - human serum albumin KW - leukotriene KW - leukotriene A(4) KW - leukotriene B(4) KW - liquid-liquid extraction KW - low QC KW - low limit of quantitation KW - mass spectrometry KW - methyl-t-butyl ether KW - mid QC KW - multiple reaction monitoring KW - phosphate buffered saline KW - quality control KW - ultra high pressure liquid chromatography SP - 59 EP - 65 JF - Journal of chromatography. B, Analytical technologies in the biomedical and life sciences JO - J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. VL - 932 N2 - Leukotriene B4 (LTB4) is a potent mediator of inflammation and has been recognized as an important target for therapeutic intervention for treatment of diseases such as asthma. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of LTB4 in human sputum as a biomarker for LTB4 biosynthesis inhibition. A fit-for-purpose strategy for method development, assay qualification, and study support was adopted for this biomarker project. A surrogate matrix (protein buffer) was used for preparation of calibration samples and certain levels of quality control (QC) samples to avoid interference from endogenous analyte, while the low QC was prepared in authentic matrix, human sputum. The analytical methodology utilized a liquid-liquid extraction procedure in 96-well plate format. Chromatographic separation was achieved with a reversed-phase ultra high pressure liquid chromatography (UPLC) column using gradient elution, and the run time was 4.5min per sample. The lower limit of quantitation (LLOQ) was 0.2ng/mL, and the calibration curve range was 0.2-20ng/mL. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (6h), freeze-thaw stability (3 cycles at -20°C), and autosampler stability (97h at ambient temperature) all met acceptance criteria. Frozen long-term stability for 166 days at -20°C in sputum did not meet acceptance criteria by showing only ≥75% of nominal concentration and the information was taken into consideration for study support. Two important observations in the current work were: (1) LTB4 was unstable in sputum in the presence of liquification reagent dithiothreitol (DTT). Therefore, a non-DTT treatment method for sputum processing was developed and applied to the bioanalytical assay and clinical study support; and (2) chromatographic separation of LTB4 from its three non-enzymatically derived isomers, i.e. 6-trans-LTB4, 12-epi-LTB4, and 6-trans-12-epi-LTB4, was achieved. This assay was successfully applied to a Phase II clinical study for proof-of-concept of a LTA4 hydrolase inhibitor for treatment of asthma. SN - 1873-376X UR - https://www.unboundmedicine.com/medline/citation/23831697/Quantitation_of_leukotriene_B_4__in_human_sputum_as_a_biomarker_using_UPLC_MS/MS_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1570-0232(13)00324-3 DB - PRIME DP - Unbound Medicine ER -