TY - JOUR
T1 - Quantification of nine antiretroviral drugs in cerebrospinal fluid
T2 - An approach to overcome sample collection tube adsorption
AU - Mykris, Timothy M.
AU - Weinhold, Jonathan
AU - Winchester, Lee C.
AU - Scarsi, Kimberly K.
AU - Fletcher, Courtney V.
AU - Podany, Anthony T.
AU - Avedissian, Sean N.
N1 - Publisher Copyright:
© 2023
PY - 2023/7/1
Y1 - 2023/7/1
N2 - A highly sensitive LC-MS/MS methods were developed and validated to quantify nine antiretrovirals (atazanavir [ATV], tenofovir [TFV], emtricitabine [FTC], darunavir [DRV], dolutegravir [DTG], efavirenz [EFV], lamivudine [3TC], raltegravir [RAL], and ritonavir [RTV]) in human cerebral spinal fluid (CSF). The approach remedies adsorption issues caused by polypropylene based sample collection tubes. 1% ammonium hydroxide in methanol was added in an amount equal to the volume of each quality control (QC) or patient sample. Protein precipitation was utilized with a CSF sample volume of 100 μL and a 100 μL of methanol:ACN and vortexed. Chromatographic separation was achieved with a 3 × 100 ACE® C18 column for ATV, DRV, DTG, EFV, RTV and RAL, and a 2 × 100 Polar RP column for TFV/FTC/3TC. Mobile phase was methanol:water:formic acid (70:30:0.1, v/v/v) for ATV, DRV, DTG, EFV and RTV (10 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (35:65:0.1, v/v/v) for RAL (50 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (2:98:0.1, v/v/v) for TFV, FTC and 3TC (50 uL injection, flow rate: 0.35 mL/min). Column temperature was 40° C across all assays. The mass spectrometer was operated in positive, multiple-reaction-monitoring (MRM) mode with electrospray ionization (ESI) for all analytes with the exception of EFV, which was operated in negative, MRM mode with ESI. The assay was linear over the calibration range of 1 to 250 ng/mL for all analytes. The addition of 1% ammonium hydroxide in sample tubes overcame up to 44% negative bias in QC samples and allowed the methods to meet full validation criteria.
AB - A highly sensitive LC-MS/MS methods were developed and validated to quantify nine antiretrovirals (atazanavir [ATV], tenofovir [TFV], emtricitabine [FTC], darunavir [DRV], dolutegravir [DTG], efavirenz [EFV], lamivudine [3TC], raltegravir [RAL], and ritonavir [RTV]) in human cerebral spinal fluid (CSF). The approach remedies adsorption issues caused by polypropylene based sample collection tubes. 1% ammonium hydroxide in methanol was added in an amount equal to the volume of each quality control (QC) or patient sample. Protein precipitation was utilized with a CSF sample volume of 100 μL and a 100 μL of methanol:ACN and vortexed. Chromatographic separation was achieved with a 3 × 100 ACE® C18 column for ATV, DRV, DTG, EFV, RTV and RAL, and a 2 × 100 Polar RP column for TFV/FTC/3TC. Mobile phase was methanol:water:formic acid (70:30:0.1, v/v/v) for ATV, DRV, DTG, EFV and RTV (10 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (35:65:0.1, v/v/v) for RAL (50 uL injection, flow rate: 1.00 mL/min), ACN:water:formic acid (2:98:0.1, v/v/v) for TFV, FTC and 3TC (50 uL injection, flow rate: 0.35 mL/min). Column temperature was 40° C across all assays. The mass spectrometer was operated in positive, multiple-reaction-monitoring (MRM) mode with electrospray ionization (ESI) for all analytes with the exception of EFV, which was operated in negative, MRM mode with ESI. The assay was linear over the calibration range of 1 to 250 ng/mL for all analytes. The addition of 1% ammonium hydroxide in sample tubes overcame up to 44% negative bias in QC samples and allowed the methods to meet full validation criteria.
KW - Adsorption
KW - Antiretroviral
KW - Cerebrospinal fluid
KW - LC-MS/MS
KW - Nonspecific binding
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UR - http://www.scopus.com/inward/citedby.url?scp=85164215450&partnerID=8YFLogxK
U2 - 10.1016/j.jchromb.2023.123810
DO - 10.1016/j.jchromb.2023.123810
M3 - Article
C2 - 37429153
AN - SCOPUS:85164215450
SN - 1570-0232
VL - 1227
JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
M1 - 123810
ER -