Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC–ESI–MS/MS: An application for pharmacokinetic studies
Yashpal S Chhonker, Richard L Sleightholm, Jing Li, David Oupický, Pharm. D Daryl J Murry
Journal of Chromatography B, doi:10.1016/j.jchromb.2017.11.026
Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C 18 (50 × 4.6 mm, 3μ) column, with gradient elution using 0.2 % formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5 mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1-2000 ng/mL for all analytes with a correlation coefficient (R 2 ) of 0.998 or better. The within-and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites.
Author Manuscript Chhonker et al. Page 17
Table 1 Summary of MS/MS parameters: precursor ion, fragment ions, voltage potential (Q1), collision energy (CE) and voltage potential (Q3) for analytes. Table 3 Mean extraction recoveries of the HCQ, DCQ, BDCQ and DHCQ from mouse blood and tissues
References
Brocks, Skeith, Johnston, Emamibafrani, Davis et al., Hematologic disposition of hydroxychloroquine enantiomers, The Journal of Clinical Pharmacology
Browning, Pharmacology of Chloroquine and Hydroxychloroquine, Hydroxychloroquine and Chloroquine Retinopathy
Chhonker, Page 22 Table 6 Blood and tissues concentration (ng/mL) and tissue to blood concentration ratio (Kp) of HCQ, DCQ, BDCQ and DHCQ after 5 mg/kg Intra venous administration of HCQ (mean ± SD, n=5)
Costedoat-Chalumeau, Amoura, Hulot, Hammoud, Aymard et al., Low blood concentration of hydroxychloroquine is a marker for and predictor of disease exacerbations in patients with systemic lupus erythematosus, Arthritis & Rheumatology
Costedoat-Chalumeau, Galicier, Aumaître, Francès, Guern et al., Hydroxychloroquine in systemic lupus erythematosus: results of a French multicentre controlled trial (PLUS Study), Annals of the Rheumatic Diseases
Fan, -W, Ma, -X, Chen, Yang et al., Pharmacokinetics and Bioequivalence Study of Hydroxychloroquine Sulfate Tablets in Chinese Healthy Volunteers by LC-MS/MS, Rheumatology and Therapy
Food, Drug Administration Centre for Drug Evaluation and Research (FDA)
Füzéry, Breaud, Emezienna, Schools, A rapid and reliable method for the quantitation of hydroxychloroquine in serum using turbulent flow liquid chromatography-tandem mass spectrometry, Clinica Chimica Acta
Goldberg, Supko, Neal, Muzikansky, Digumarthy et al., A Phase I Study of Erlotinib and Hydroxychloroquine in Advanced Non-Small-Cell Lung Cancer, Journal of Thoracic Oncology
Hopfgartner, Bourgogne, Quantitative high-throughput analysis of drugs in biological matrices by mass spectrometry, Mass spectrometry reviews
Lim, Im, Cho, Bae, Klein et al., Pharmacokinetics of Hydroxychloroquine and Its Clinical Implications in Chemoprophylaxis against Malaria Caused by Plasmodium vivax, Antimicrobial Agents and Chemotherapy
Mezzaroba, Zorzet, Secco, Biffi, Tripodo et al., New Potential Therapeutic Approach for the Treatment of B-Cell Malignancies Using Chlorambucil/Hydroxychloroquine-Loaded Anti-CD20 Nanoparticles, PLOS ONE
Munster, Gibbs, Shen, Baethge, Botstein et al., Hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis, Arthritis & Rheumatology
Qu, Noe, Gaelle, Breaud, Vidal et al., Development and validation of a clinical HPLC method for the quantification of hydroxychloroquine and its metabolites in whole blood, Future Science OA
Rolain, Colson, Raoult, Recycling of chloroquine and its hydroxyl analogue to face bacterial, fungal and viral infections in the 21st century, International Journal of Antimicrobial Agents
Skopp, Pötsch, Mauden, Richter, Partition coefficient, blood to plasma ratio, protein binding and short-term stability of 11-nor-Δ9-carboxy tetrahydrocannabinol glucuronide, Forensic Science International
Soichot, Mégarbane, Houzé, Chevillard, Fonsart et al., Development, validation and clinical application of a LC-MS/MS method for the simultaneous quantification of hydroxychloroquine and its active metabolites in human whole blood, J Pharm Biomed Anal
Tett, Cutler, Brown, High-performance liquid chromatographic assay for hydroxychloroquine and metabolites in blood and plasma, using a stationary phase of poly(styrene divinylbenzene) and a mobile phase at ph 11, with fluorimetric detection, Journal of Chromatography B: Biomedical Sciences and Applications
Van Dongen, Niessen, LC-MS systems for quantitative bioanalysis
Wang, Ong, Chin, Thuya, Wan et al., Method development and validation for rapid quantification of hydroxychloroquine in human blood using liquid chromatography-tandem mass spectrometry, J Pharm Biomed Anal
Williams, Patchen, Churchill, Analysis of blood and urine samples for hydroxychloroquine and three major metabolites by high-performance liquid chromatography with fluorescence detection, Journal of Chromatography B: Biomedical Sciences and Applications
Yang, Wang, Contino, Liesa, Sahin et al., Pancreatic cancers require autophagy for tumor growth, Genes Dev
Yang, Wang, Hou, Tung, Chiu et al., Blockade of autophagy reduces pancreatic cancer stem cell activity and potentiates the tumoricidal effect of gemcitabine, Mol Cancer
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'multicentre controlled trial (PLUS Study)',
'volume': '72',
'author': 'Costedoat-Chalumeau',
'year': '2013',
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'doi-asserted-by': 'crossref',
'first-page': '297',
'DOI': '10.1016/j.ijantimicag.2007.05.015',
'article-title': 'Recycling of chloroquine and its hydroxyl analogue to face bacterial, '
'fungal and viral infections in the 21st century',
'volume': '30',
'author': 'Rolain',
'year': '2007',
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'doi-asserted-by': 'crossref',
'first-page': '717',
'DOI': '10.1101/gad.2016111',
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'year': '2011',
'journal-title': 'Genes Dev.'},
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'first-page': '1',
'DOI': '10.1186/s12943-015-0449-3',
'article-title': 'Blockade of autophagy reduces pancreatic cancer stem cell activity and '
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'volume': '14',
'author': 'Yang',
'year': '2015',
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'first-page': 'e74216',
'DOI': '10.1371/journal.pone.0074216',
'article-title': 'New potential therapeutic approach for the treatment of B-Cell '
'malignancies using chlorambucil/hydroxychloroquine-loaded anti-CD20 '
'nanoparticles',
'volume': '8',
'author': 'Mezzaroba',
'year': '2013',
'journal-title': 'PLoS One'},
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'first-page': '1602',
'DOI': '10.1097/JTO.0b013e318262de4a',
'article-title': 'A phase I study of erlotinib and hydroxychloroquine in advanced '
'non–small-cell lung cancer',
'volume': '7',
'author': 'Goldberg',
'year': '2012',
'journal-title': 'J. Thorac. Oncol.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0045',
'doi-asserted-by': 'crossref',
'first-page': '1460',
'DOI': '10.1002/art.10307',
'article-title': 'Hydroxychloroquine concentration–response relationships in patients '
'with rheumatoid arthritis',
'volume': '46',
'author': 'Munster',
'year': '2002',
'journal-title': 'Arthritis Rheumatol.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0050',
'doi-asserted-by': 'crossref',
'first-page': '1088',
'DOI': '10.1002/j.1552-4604.1994.tb01986.x',
'article-title': 'Hematologic disposition of hydroxychloroquine enantiomers',
'volume': '34',
'author': 'Brocks',
'year': '1994',
'journal-title': 'J. Clin. Pharmacol.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0055',
'doi-asserted-by': 'crossref',
'first-page': '241',
'DOI': '10.1016/S0378-4347(00)82024-1',
'article-title': 'High-performance liquid chromatographic assay for hydroxychloroquine '
'and metabolites in blood and plasma using a stationary phase of '
'poly(styrene divinylbenzene) and a mobile phase at ph 11, with '
'fluorimetric detection',
'volume': '344',
'author': 'Tett',
'year': '1985',
'journal-title': 'J. Chromatogr. B: Biomed. Sci. Appl.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0060',
'doi-asserted-by': 'crossref',
'first-page': '197',
'DOI': '10.1016/S0378-4347(00)80598-8',
'article-title': 'Analysis of blood and urine samples for hydroxychloroquine and three '
'major metabolites by high-performance liquid chromatography with '
'fluorescence detection',
'volume': '433',
'author': 'Williams',
'year': '1988',
'journal-title': 'J. Chromatogr. B: Biomed. Sci. Appl.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0065',
'doi-asserted-by': 'crossref',
'first-page': '3284',
'DOI': '10.1002/art.22156',
'article-title': 'Low blood concentration of hydroxychloroquine is a marker for and '
'predictor of disease exacerbations in patients with systemic lupus '
'erythematosus',
'volume': '54',
'author': 'Costedoat-Chalumeau',
'year': '2006',
'journal-title': 'Arthritis Rheumatol.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0070',
'doi-asserted-by': 'crossref',
'DOI': '10.4155/fso.15.24',
'article-title': 'Development and validation of a clinical HPLC method for the '
'quantification of hydroxychloroquine and its metabolites in whole blood',
'volume': '1',
'author': 'Qu',
'year': '2015',
'journal-title': 'Future Sci. OA'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0075',
'doi-asserted-by': 'crossref',
'first-page': '195',
'DOI': '10.1002/mas.10050',
'article-title': 'Quantitative high-throughput analysis of drugs in biological matrices '
'by mass spectrometry',
'volume': '22',
'author': 'Hopfgartner',
'year': '2003',
'journal-title': 'Mass Spectrom. Rev.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0080',
'doi-asserted-by': 'crossref',
'unstructured': 'W.D. van Dongen, W.M. Niessen, LC–MS systems for quantitative '
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'DOI': '10.4155/bio.12.221'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0085',
'doi-asserted-by': 'crossref',
'first-page': '79',
'DOI': '10.1016/j.cca.2013.02.018',
'article-title': 'A rapid and reliable method for the quantitation of hydroxychloroquine '
'in serum using turbulent flow liquid chromatography-tandem mass '
'spectrometry',
'volume': '421',
'author': 'Füzéry',
'year': '2013',
'journal-title': 'Clin. Chim. Acta'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0090',
'doi-asserted-by': 'crossref',
'first-page': '86',
'DOI': '10.1016/j.jpba.2011.11.034',
'article-title': 'Method development and validation for rapid quantification of '
'hydroxychloroquine in human blood using liquid chromatography–tandem '
'mass spectrometry',
'volume': '61',
'author': 'Wang',
'year': '2012',
'journal-title': 'J. Pharm. Biomed. Anal.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0095',
'doi-asserted-by': 'crossref',
'first-page': '131',
'DOI': '10.1016/j.jpba.2014.07.009',
'article-title': 'Development, validation and clinical application of a LC-MS/MS method '
'for the simultaneous quantification of hydroxychloroquine and its '
'active metabolites in human whole blood',
'volume': '100',
'author': 'Soichot',
'year': '2014',
'journal-title': 'J. Pharm. Biomed. Anal.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0100',
'doi-asserted-by': 'crossref',
'first-page': '183',
'DOI': '10.1007/s40744-015-0012-0',
'article-title': 'Pharmacokinetics and bioequivalence study of hydroxychloroquine sulfate '
'tablets in Chinese healthy volunteers by LC–MS/MS',
'volume': '2',
'author': 'Fan',
'year': '2015',
'journal-title': 'Rheumatol. Ther.'},
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'doi-asserted-by': 'crossref',
'first-page': '17',
'DOI': '10.1016/S0379-0738(02)00023-3',
'article-title': 'Partition coefficient, blood to plasma ratio, protein binding and '
'short-term stability of 11-nor-Δ9-carboxy tetrahydrocannabinol '
'glucuronide',
'volume': '126',
'author': 'Skopp',
'year': '2002',
'journal-title': 'Forensic Sci. Int.'},
{ 'key': '10.1016/j.jchromb.2017.11.026_bib0110',
'series-title': 'Guidance for Industry-Bioanalytical Method Validation',
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