Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes
Abstract
:1. Introduction
2. Results and Discussion
2.1. UHPLC-MS/MS Method Development in Reversed Phase Mode
2.2. UHPLC-MS/MS Method Development in HILIC Mode
2.3. Optimization of µ-SPE-PT Sample Preparation
2.4. Method Validation
2.5. Comparison of the HILIC and RP Modes
3. Materials and Methods
3.1. Chemicals
3.2. Instrumentation and UHPLC-MS/MS Analysis
3.3. Stock Solutions of Reference Standards
3.4. Biological Experiments to Evaluate Drug-Drug Interactions
3.5. Sample Preparation
3.6. Method Validation
4. Conclusions
Author Contributions
Funding
Conflicts of Interest
Sample Availability
References
- Nováková, L.; Pavlík, J.; Chrenková, L.; Martinec, O.; Červený, L. Current antiviral drugs and their analysis in biological materials—Part I: Antivirals against respiratory and herpes viruses. J. Pharm. Biomed. Anal. 2018, 147, 400–416. [Google Scholar] [CrossRef] [PubMed]
- De Clercq, E.; Li, G. Approved Antiviral Drugs over the Past 50 Years. Clin. Microbiol. Rev. 2016, 29, 695–747. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Li, G.; Xu, M.; Yue, T.; Gu, W.; Tan, L. Life-long passion for antiviral research and drug development: 80th birthday of Prof. Dr. Erik De Clercq. Biochem. Pharmacol. 2021, 185, 114485. [Google Scholar] [CrossRef] [PubMed]
- FDA. New Drugs at FDA: CDER’s New Molecular Entities and New Therapeutic Biological Products. Available online: https://www.fda.gov/drugs/development-approval-process-drugs/new-drugs-fda-cders-new-molecular-entities-and-new-therapeutic-biological-products (accessed on 1 March 2021).
- Tomić, D.; Davidović, D.; Szasz, A.M.; Rezeli, M.; Pirkić, B.; Petrik, J.; Vrca, V.B.; Janđel, V.; Lipić, T.; Skala, K.; et al. The screening and evaluation of potential clinically significant HIV drug combinations against the SARS-CoV-2 virus. Inform. Med. Unlocked 2021, 100529. [Google Scholar] [CrossRef]
- Baby, K.; Maity, S.; Mehta, C.H.; Suresh, A.; Nayak, U.Y.; Nayak, Y. SARS-CoV-2 entry inhibitors by dual targeting TRPRSS2 and ACE2: An in silico drug repurposing study. Eur. J. Pharmacol. 2021, 896, 173922–173933. [Google Scholar] [CrossRef]
- Fritz, A.; Busch, D.; Lapczuk, J.; Ostrowski, M.; Drozdzik, M.; Oswald, S. Expression of clinically relevant drug-metabolizing enzymes along the human intestine and their correlation to drug transporters and nuclear receptors: An intra-subject analysis. Basic Clin. Pharmacol. Toxicol. 2019, 124, 245–255. [Google Scholar] [CrossRef]
- Rodrigues, M.C.S.; de Oliveira, C. Drug-drug interactions and adverse drug reactions in polypharmacy among older adults: An integrative review. Rev. Latino-Am. Enfermagem 2016, 24, 17. [Google Scholar] [CrossRef]
- Nováková, L.; Pavlík, J.; Chrenková, L.; Martinec, O.; Červený, L. Current antiviral drugs and their analysis in biological materials – Part II: Antivirals against hepatitis and HIV viruses. J. Pharm. Biomed. Anal. 2018, 147, 378–399. [Google Scholar] [CrossRef]
- Martinec, O.; Huliciak, M.; Staud, F.; Cecka, F.; Vokral, I.; Cerveny, L. Anti-HIV and Anti-Hepatitis C Virus Drugs Inhibit P-Glycoprotein Efflux Activity in Caco-2 Cells and Precision-Cut Rat and Human Intestinal Slices. Antimicrob. Agents Chemother. 2019, 63, 00910-19. [Google Scholar] [CrossRef] [Green Version]
- FDA. In Vitro Drug Interaction Studies-Cytochrome P450 Enzyme-and Transporter-Mediated Drug Interactions Guidance for Industry. Available online: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/vitro-drug-interaction-studies-cytochrome-p450-enzyme-and-transporter-mediated-drug-interactions (accessed on 6 April 2021).
- EMA. Guideline on the Investigation of Drug Interactions—Revision 1, CPMP/EWP/560/95/Rev.1 Corr.2. Available online: www.ema.europa.eu/contact.2012. (accessed on 6 April 2021).
- Acquavia, M.A.; Foti, L.; Pascale, R.; Nicolò, A.; Brancaleone, V.; Cataldi, T.R.; Martelli, G.; Scrano, L.; Bianco, G. Detection and quantification of Covid-19 antiviral drugs in biological fluids and tissues. Talanta 2021, 224, 121862. [Google Scholar] [CrossRef]
- Else, L.; Watson, V.; Tjia, J.; Hughes, A.; Siccardi, M.; Khoo, S.; Back, D. Validation of a rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) assay for the simultaneous determination of existing and new antiretroviral compounds. J. Chromatogr. B 2010, 878, 1455–1465. [Google Scholar] [CrossRef]
- D’Avolio, A.; Simiele, M.; Siccardi, M.; Baietto, L.; Sciandra, M.; Oddone, V.; Stefani, F.R.; Agati, S.; Cusato, J.; Bonora, S.; et al. A HPLC-MS method for the simultaneous quantification of fourteen antiretroviral agents in peripheral blood mon-onuclear cell of HIV infected patients optimized using medium corpuscular volume evaluation. J. Pharm. Biomed. Anal. 2011, 54, 779–788. [Google Scholar] [CrossRef]
- Djerada, Z.; Feliu, C.; Tournois, C.; Vautier, D.; Binet, L.; Robinet, A.; Marty, H.; Gozalo, C.; Lamiable, D.; Millart, H. Validation of a fast method for quantitative analysis of elvitegravir, raltegravir, maraviroc, etravirine, tenofovir, boceprevir and 10 other antiretroviral agents in human plasma samples with a new UPLC-MS/MS technology. J. Pharm. Biomed. Anal. 2013, 86, 100–111. [Google Scholar] [CrossRef]
- Watanabe, K.; Varesio, E.; Hopfgartner, G. Parallel ultra high-pressure liquid chromatography–mass spectrometry for the quantification of HIV protease inhibitors using dried spot sample collection format. J. Chromatogr. B 2014, 965, 244–253. [Google Scholar] [CrossRef]
- Marzinke, M.A.; Breaud, A.; Parsons, T.L.; Cohen, M.S.; Piwowar-Manning, E.; Eshleman, S.H.; Clarke, W. The development and validation of a method using high-resolution mass spectrometry (HRMS) for the qualitative detection of antiretroviral agents in human blood. Clin. Chim. Acta 2014, 433, 157–168. [Google Scholar] [CrossRef] [Green Version]
- Ariaudo, A.; Favata, F.; De Nicolò, A.; Simiele, M.; Paglietti, L.; Boglione, L.; Cardellino, C.S.; Carcieri, C.; Di Perri, G.; D’Avolio, A. A UHPLC-MS/MS method for the quantification of direct antiviral agents simeprevir, daclatasvir, ledipasvir, sofos-buvir/GS-331007, dasabuvir, ombitasvir and paritaprevir, together with ritonavir, in human plasma. J. Pharm. Biomed. Anal. 2016, 125, 369–375. [Google Scholar] [CrossRef]
- Simiele, M.; Ariaudo, A.; De Nicolò, A.; Favata, F.; Ferrante, M.; Carcieri, C.; Bonora, S.; Di Perri, G.; D’Avolio, A. UPLC–MS/MS method for the simultaneous quantification of three new antiretroviral drugs, dolutegravir, elvitegravir and rilpivirine, and other thirteen antiretroviral agents plus cobicistat and ritonavir boosters in human plasma. J. Pharm. Biomed. Anal. 2017, 138, 223–230. [Google Scholar] [CrossRef]
- Conti, M.; Cavedagna, T.M.; Ramazzotti, E.; Mancini, R.; Calza, L.; Rinaldi, M.; Badia, L.; Guardigni, V.; Viale, P.; Verucchi, G. Multiplexed therapeutic drug monitoring (TDM) of antiviral drugs by LC–MS/MS. Clin. Mass Spectrom. 2018, 7, 6–17. [Google Scholar] [CrossRef]
- Daskapan, A.; van Hateren, K.; Stienstra, Y.; Kosterink, J.; van der Werf, T.; Touw, D.; Alffenaar, J.-W. Development and validation of a bioanalytical method for the simultaneous determination of 14 antiretroviral drugs using liquid chroma-tography-tandem mass spectrometry. J. Appl. Bioanal. 2018, 4, 37–50. [Google Scholar] [CrossRef] [Green Version]
- van Seyen, M.; de Graaff Teulen, M.J.A.; van Erp, N.P.; Burger, D.M. Quantification of second generation direct-acting antivirals daclatasvir, elbasvir, grazoprevir, ledipasvir, simeprevir, sofosbuvir and velpatasvir in human plasma by UPLC-MS/MS. J. Chromatogr. B 2019, 1110, 15–24. [Google Scholar] [CrossRef]
- Gouget, H.; Noé, G.; Barrail-Tran, A.; Furlan, V. UPLC–MS/MS method for the simultaneous quantification of bictegravir and 13 others antiretroviral drugs plus cobicistat and ritonavir boosters in human plasma. J. Pharm. Biomed. Anal. 2020, 181, 113057. [Google Scholar] [CrossRef] [PubMed]
- Zheng, Y.; Aboura, R.; Boujaafar, S.; Lui, G.; Hirt, D.; Bouazza, N.; Foissac, F.; Treluyer, J.-M.; Benaboud, S.; Gana, I. HPLC-MS/MS method for the simultaneous quantification of dolutegravir, elvitegravir, rilpivirine, darunavir, ritonavir, raltegravir and raltegravir-β-d-glucuronide in human plasma. J. Pharm. Biomed. Anal. 2020, 182, 113119. [Google Scholar] [CrossRef] [PubMed]
- Habler, K.; Brügel, M.; Teupser, D.; Liebchen, U.; Scharf, C.; Schönermarck, U.; Vogeser, M.; Paal, M. Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ri-tonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS in human serum. J. Pharm. Biomed. Anal. 2021, 196, 113935–113943. [Google Scholar] [CrossRef] [PubMed]
- Niessen, W. Tandem mass spectrometry of small-molecule antiviral drugs: 1. HIV-related antivirals. Int. J. Mass Spectrom. 2020, 455, 116370. [Google Scholar] [CrossRef]
- Niessen, W. Tandem mass spectrometry of small-molecule antiviral drugs: 2. hepatitis-related antivirals. Int. J. Mass Spectrom. 2020, 455, 116371. [Google Scholar] [CrossRef]
- Niessen, W. Tandem mass spectrometry of small-molecule antiviral drugs: 3. antiviral agents against herpes, influenza and other viral infections. Int. J. Mass Spectrom. 2020, 455, 116377. [Google Scholar] [CrossRef]
- Nováková, L.; Havlíková, L.; Vlčková, H. Hydrophilic interaction chromatography of polar and ionizable compounds by UHPLC. TrAC Trends Anal. Chem. 2014, 63, 55–64. [Google Scholar] [CrossRef]
- Hsiao, J.J.; Potter, O.G.; Chu, T.-W.; Yin, H. Improved LC/MS Methods for the Analysis of Metal-Sensitive Analytes Using Medronic Acid as a Mobile Phase Additive. Anal. Chem. 2018, 90, 9457–9464. [Google Scholar] [CrossRef]
- Vlčková, H.; Pilařová, V.; Novák, O.; Solich, P.; Nováková, L. Micro-SPE in pipette tips as a tool for analysis of small-molecule drugs in serum. Bioanal. 2017, 9, 887–901. [Google Scholar] [CrossRef]
- Ocque, A.J.; Hagler, C.E.; Morse, G.D.; Letendre, S.L.; Ma, Q. Development and validation of an LC–MS/MS assay for tenofovir and tenofovir alafenamide in human plasma and cerebrospinal fluid. J. Pharm. Biomed. Anal. 2018, 156, 163–169. [Google Scholar] [CrossRef]
- Yadav, M.; Mishra, T.; Singhal, P.; Goswami, S.; Shrivastav, P.S. Rapid and specific liquid chromatographic tandem mass spectrometric determination of tenofovir in human plasma and its fragmentation study. J. Chromatogr. Sci. 2009, 47, 140–148. [Google Scholar] [CrossRef]
- International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). M10: Bioanalytical Method Validation. Available online: https://www.ema.europa.eu/en/ich-m10-bioanalytical-method-validation (accessed on 6 April 2021).
- European Medicines Agency. Committee for Medicinal Products for Human Use, Guidelines on Validation of Bioanalytical Methods (draft), EMA/CMP/EWP/192217/200, London, UK, 2011. Available online: https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method-validation_en.pdf (accessed on 6 April 2021).
- Hubatsch, I.; E Ragnarsson, E.G.; Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2007, 2, 2111–2119. [Google Scholar] [CrossRef]
Analyte | Abbreviation | Exact Mass (Da) | logP | pKa (Acidic) | pKa (Basic) | Acid-Base Properties | tR in RP (min) | tR in HILIC (min) | Precursor Ion Type | Precursor m/z | Fragment m/z | CV a in RP (V) | CV in HILIC (V) | CE b (eV) |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
abacavir | ABA | 286.1542 | 0.39 | 15.43 | 5.80 | basic | 1.63 | 3.77 | [M + H]+ | 287.2 | 191.0 | 15 | 15 | 20 |
atazanavir | ATA | 704.3897 | 4.54 | 11.92 | 4.42 | basic | 3.60 | 2.05 | [M + H]+ | 705.3 | 168.0 | 25 | 25 | 50 |
boceprevir | BOC | 519.3421 | 1.70 | 12.44 | −0.92 | neutral | 4.10 | 1.62 | [M + H]+ | 520.3 | 308.1 | 35 | 35 | 25 |
daclatasvir | DAC | 738.3853 | 5.11 | 12.47 | 5.40 | basic | 2.25 | 4.21 | [M + H]+ | 739.2 | 565.1 | 10 | 10 | 40 |
didanosine | DID | 236.0909 | −0.35 | 10.94 | 2.76 | neutral | 1.51 | 3.34 | [M − H]− | 235.0 | 135.0 | 40 | 55 | 20 |
doravirine | DOR | 425.0503 | 2.23 | 9.66 | n/a | acidic | 3.60 | 2.16 | [M + H]+ | 426.0 | 314.9 | 40 | 55 | 20 |
efavirenz | EFA | 315.0274 | 4.46 | 12.52 | −1.49 | neutral | 4.60 | 1.42 | [M + H]+ | 316.0 | 243.9 | 20 | 35 | 15 |
glecaprevir | GLE | 838.2983 | 3.95 | 3.74 | −1.20 | acidic | 5.01 | 1.42 | [M + H]+ | 839.1 | 819.1 | 15 | 15 | 15 |
ledipasvir | LED | 888.4134 | 6.71 | 11.22 | 5.32 | basic | 3.20 | 2.92 | [M + 2H]2+ | 445.4 | 130.0 | 10 | 10 | 25 |
lopinavir | LOP | 628.3625 | 4.69 | 13.39 | −1.55 | neutral | 4.36 | 2.50 | [M + H]+ | 629.3 | 155.0 | 15 | 15 | 40 |
maraviroc | MAR | 513.3279 | 3.63 | 13.98 | 9.35 | basic | 2.25 | 4.99 | [M + H]+ | 514.2 | 280.1 | 15 | 15 | 30 |
rilpivirine | RIL | 366.1593 | 5.47 | 11.43 | 4.44 | basic | 2.95 | 1.71 | [M − H]− | 365.1 | 141.9 | 55 | 70 | 25 |
ritonavir | RIT | 720.3128 | 5.22 | 13.68 | 2.84 | neutral | 4.26 | 2.53 | [M + H]+ | 721.2 | 296.1 | 15 | 15 | 20 |
saquinavir | SAQ | 670.3843 | 3.16 | 13.61 | 8.47 | basic | 2.95 | 4.18 | [M + H]+ | 671.3 | 570.1 | 55 | 55 | 30 |
sofosbuvir | SOF | 529.1625 | 1.28 | 9.70 | n/a | acidic | 3.23 | 1.91 | [M + H]+ | 530.1 | 243.0 | 15 | 15 | 20 |
tenofovir | TEN | 287.0783 | −3.44 | 1.35 | 3.74 | acidic | 0.96 | 5.65 | [M + H]+ | 288.1 | 176.1 | 15 | 15 | 25 |
tenofovir alafenamide | TNA | 476.1937 | 1.88 | 11.36 | 3.74 | basic | 2.57 | 3.39 | [M + H]+ | 477.1 | 270.0 | 15 | 15 | 30 |
tenofovir disoproxil | TDF | 519.1730 | 2.65 | n/a | 3.74 | basic | 2.76 | 3.13 | [M + H]+ | 520.1 | 270.0 | 15 | 15 | 25 |
tenofovir monoester | MONO | 403.1257 | −1.70 | 1.07 | 3.74 | acidic | 1.99 | 4.64 | [M + H]+ | 404.1 | 270.0 | 15 | 45 | 20 |
velpatasvir | VEL | 882.4065 | 5.11 | 11.71 | 5.36 | basic | 2.95 | 3.48 | [M + 2H]2+ | 442.4 | 405.0 | 20 | 10 | 25 |
zidovudine | ZID | 267.0968 | −0.30 | 4.22 | n/a | acidic | 2.19 | 1.85 | [M − H]− | 266.0 | 223.0 | 30 | 35 | 10 |
LOD (ng/mL) | LLOQ (ng/mL) | ULOQ (ng/mL) | r2 pH 7.4 pH 6.5 | Concentration Levels (ng/mL) (n = 5) | Accuracy (%) pH 7.4 Buffer pH 6.5 Buffer | Precision (% RSD) pH 7.4 Buffer pH 6.5 Buffer | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
L1 | L2 | L3 | L4 | L1 | L2 | L3 | L4 | L1 | L2 | L3 | L4 | |||||
ABA | 0.03 | 0.1 | 100 | 0.9991 | 0.1 | 0.2 | 20 | 50 | 16.3 | 4.7 | −0.5 | 0.6 | 1.3 | 1.2 | 5.0 | 4.4 |
0.9995 | −1.4 | 6.9 | 7.9 | 3.1 | 2.7 | 5.3 | 4.9 | 2.1 | ||||||||
ATA | 0.15 | 0.5 | 100 | 0.9981 | 0.5 | 1 | 20 | 50 | 1.9 | −9.1 | 1.7 | 7.1 | 2.7 | 3.0 | 2.2 | 3.8 |
0.06 | 0.2 | 100 | 0.9988 | 12.4 | 13.3 | 14.8 | 11.8 | 2.0 | 1.0 | 3.3 | 2.4 | |||||
BOC | 0.6 | 2 | 1000 | 0.9996 | 2 | 5 | 200 | 500 | 6.7 | 4.3 | −14.1 | −14.7 | 4.7 | 3.1 | 1.8 | 2.3 |
0.3 | 1 | 1000 | 0.9996 | 1 | 2 | 4.5 | 10.5 | −3.3 | −8.9 | 4.6 | 5.0 | 1.9 | 2.0 | |||
DAC | 1.5 | 5 | 1000 | 0.9960 | 5 | 10 | 100 | 200 | 18.7 | 10.7 | 10.2 | 14.6 | 12.8 | 13.7 | 13.8 | 5.9 |
0.9970 | 200 | 500 | 45.9 | 43.3 | −27.6 | −17.8 | 13.5 | 11.6 | 10.0 | 2.9 | ||||||
DID | 6 | 20 | 1000 | 0.9990 | 20 | 50 | 100 | 500 | 5.8 | −1.7 | −4.0 | −4.6 | 4.8 | 6.0 | 6.2 | 9.2 |
0.9981 | 14.1 | 13.5 | −1.7 | −12.8 | 6.1 | 5.4 | 11.1 | 5.0 | ||||||||
DOR | 0.15 | 0.5 | 100 | 0.9994 | 0.5 | 1 | 20 | 50 | −2.5 | −7.8 | −3.8 | −6.4 | 6.1 | 6.4 | 2.5 | 3.2 |
0.06 | 0.2 | 100 | 0.9992 | 0.2 | 0.5 | 3.1 | 1.5 | 13.5 | 7.3 | 8.5 | 2.8 | 2.9 | 1.6 | |||
EFA | 0.6 | 2 | 1000 | 0.9989 | 2 | 5 | 10 | 50 | 10.0 | −4.2 | −4.2 | −2.6 | 1.7 | 3.3 | 4.6 | 2.8 |
0.9984 | 19.0 | 12.6 | 2.5 | −10.2 | 6.4 | 9.6 | 8.9 | 10.6 | ||||||||
GLE | 3 | 10 | 1000 | 0.9949 | 10 | 50 | 200 | 500 | −7.4 | −17.0 | −65.0 | −63.4 | 25.0 | 25.6 | 9.9 | 12.2 |
15 | 50 | 1000 | 0.9971 | 50 | 100 | −52.7 | 94.7 | 90.5 | 68.8 | 26.2 | 5.7 | 1.9 | 4.6 | |||
LED | 1.5 | 5 | 200 | 0.9991 | 5 | 10 | 100 | 200 | −2.7 | −14.8 | 0.2 | 1.6 | 7.5 | 6.1 | 8.3 | 12.6 |
0.9980 | 50 | 100 | −15.3 | 3.0 | −4.3 | 185.5 | 13.4 | 16.1 | 20.0 | 16.6 | ||||||
LOP | 0.3 | 1 | 1000 | 0.9998 | 1 | 2 | 10 | 50 | 9.5 | 0.7 | −13.6 | −14.9 | 8.6 | 6.6 | 6.1 | 2.9 |
0.9997 | 200 | 500 | 13.5 | 8.1 | 13.3 | 11.3 | 5.9 | 5.9 | 4.5 | 5.1 | ||||||
MAR | 0.3 | 1 | 100 | 0.9970 | 1 | 5 | 20 | 50 | −4.2 | −14.5 | −4.2 | −1.0 | 1.7 | 3.6 | 2.9 | 5.6 |
0.9979 | 13.3 | −6.8 | 5.3 | 3.9 | 3.5 | 2.9 | 3.6 | 3.4 | ||||||||
RIL | 0.6 | 2 | 1000 | 0.9986 | 10 | 50 | 100 | 200 | 3.6 | −6.6 | −4.6 | −6.2 | 8.1 | 7.7 | 8.8 | 2.8 |
500 | 0.9972 | 200 | 500 | 20.0 | 7.4 | 0.8 | −9.1 | 2.8 | 1.6 | 7.1 | 5.9 | |||||
RIT | 0.3 | 1 | 1000 | 0.9996 | 1 | 2 | 100 | 200 | 11.2 | 5.8 | −10.7 | −11.6 | 9.7 | 6.0 | 4.5 | 2.6 |
0.9996 | 200 | 500 | 9.6 | 9.4 | 13.0 | 9.8 | 6.5 | 4.9 | 4.3 | 3.4 | ||||||
SAQ | 0.06 | 0.2 | 100 | 0.9982 | 0.2 | 0.5 | 20 | 50 | 26.5 | 35.1 | 31.9 | 54.3 | 6.0 | 14.5 | 6.5 | 5.4 |
0.9985 | 1 | 5 | −6.0 | 9.0 | 6.7 | 10.0 | 11.8 | 10.8 | 9.8 | 13.7 | ||||||
SOF | 0.03 | 0.1 | 100 | 0.9993 | 0.1 | 0.2 | 20 | 50 | 10.4 | 5.6 | 1.2 | 2.3 | 6.4 | 2.9 | 2.0 | 3.3 |
0.06 | 0.2 | 0.9994 | 0.2 | 0.5 | 19.4 | 9.3 | 6.2 | 1.4 | 2.1 | 4.5 | 3.7 | 2.6 | ||||
TEN | 0.3 | 1 | 100 | 0.9924 | 1 | 5 | 20 | 50 | 18.1 | −13.0 | −5.1 | 9.0 | 9.7 | 6.0 | 4.5 | 2.6 |
0.9960 | 19.3 | −9.3 | 1.8 | 3.8 | 5.6 | 4.0 | 5.8 | 4.4 | ||||||||
TNA | 0.03 | 0.1 | 100 | 0.9996 | 0.1 | 0.2 | 20 | 50 | 6.8 | 2.9 | −5.4 | −2.6 | 4.8 | 4.2 | 0.8 | 2.3 |
0.9998 | 5.4 | 10.9 | 5.3 | 3.8 | 3.7 | 5.4 | 2.9 | 2.8 | ||||||||
TDF | 0.03 | 0.1 | 100 | 0.9993 | 0.1 | 0.2 | 20 | 50 | 16.3 | 3.3 | −6.0 | −4.3 | 9.6 | 9.8 | 3.5 | 3.8 |
0.9997 | 10.7 | 15.0 | 6.4 | 2.7 | 12.1 | 7.1 | 3.2 | 3.7 | ||||||||
MONO | 0.03 | 0.1 | 100 | 0.9995 | 0.1 | 0.2 | 20 | 50 | 3.0 | 8.6 | 1.3 | 2.9 | 5.1 | 6.2 | 3.5 | 3.1 |
0.9995 | 0.7 | 7.3 | 13.4 | 13.1 | 10.1 | 5.6 | 3.0 | 3.3 | ||||||||
VEL | 6 | 20 | 10000 | 0.9976 | 20 | 50 | 100 | 200 | −13.4 | −14.6 | −27.6 | −23.1 | 14.0 | 14.6 | 15.0 | 15.0 |
3 | 10 | 1000 | 0.9975 | 10 | 20 | −13.2 | −12.6 | −17.0 | −13.6 | 17.3 | 10.7 | 6.3 | 9.4 | |||
ZID | 0.3 | 1 | 1000 | 0.9986 | 1 | 2 | 200 | 500 | −15.8 | 1.1 | 12.4 | 7.4 | 17.8 | 9.8 | 5.4 | 3.5 |
0.9985 | −14.7 | 13.9 | 14.3 | 13.1 | 11.2 | 6.5 | 3.8 | 6.9 |
LOD (ng/mL) | LLOQ (ng/mL) | ULOQ (ng/mL) | r2 pH 7.4 pH 6.5 | Concentration Levels (ng/mL) (n = 5) | Accuracy (%) pH 7.4 Buffer pH 6.5 Buffer | Precision (% RSD) pH 7.4 Buffer pH 6.5 Buffer | ||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
L1 | L2 | L3 | L4 | L1 | L2 | L3 | L4 | L1 | L2 | L3 | L4 | |||||
ABA | 0.0015 | 0.005 | 20 | 0.9999 | 0.005 | 0.01 | 5 | 10 | 8.0 | 4.0 | 0.5 | −1.0 | 4.1 | 6.3 | 1.4 | 1.6 |
0.9998 | −4.0 | −5.0 | 0.1 | 0.1 | 5.7 | 3.7 | 1.7 | 2.4 | ||||||||
ATA | 0.0006 | 0.002 | 20 | 0.9998 | 0.002 | 0.005 | 5 | 10 | 10.0 | 12.0 | 4.3 | 2.4 | 12.4 | 4.0 | 1.1 | 1.3 |
0.9947 | 15.0 | −4.0 | 7.6 | 9.1 | 17.4 | 5.7 | 1.5 | 1.5 | ||||||||
BOC | 0.015 | 0.05 | 200 | 0.9998 | 0.05 | 0.1 | 50 | 100 | 10.6 | 9.8 | 1.2 | 0.7 | 2.7 | 7.7 | 1.6 | 1.8 |
0.9995 | 0.0 | −4.4 | 5.9 | −1.5 | 2.3 | 2.6 | 1.3 | 1.0 | ||||||||
DAC | 0.015 | 0.05 | 200 | 0.9997 | 0.05 | 0.1 | 50 | 100 | 2.4 | 7.9 | 0.3 | 0.7 | 2.9 | 3.9 | 2.9 | 0.6 |
0.9989 | 1.2 | 2.9 | 2.2 | 4.3 | 4.9 | 3.8 | 1.9 | 2.0 | ||||||||
DID | 0.3 | 1 | 2000 | 0.9991 | 1 | 2 | 500 | 1000 | 5.6 | −0.2 | 3.1 | 1.7 | 4.3 | 8.9 | 9.1 | 2.5 |
0.9993 | 9.8 | −3.3 | −0.5 | −10.8 | 10.0 | 6.2 | 2.6 | 3.8 | ||||||||
DOR | 0.0015 | 0.005 | 20 | 0.9998 | 0.005 | 0.01 | 5 | 10 | 2.0 | 1.0 | −0.3 | −7.5 | 8.2 | 7.3 | 1.0 | 1.0 |
0.9995 | −2.0 | −1.0 | 0.1 | −8.2 | 4.6 | 9.0 | 4.8 | 2.9 | ||||||||
EFA | 3 | 10 | 2000 | 0.9995 | 10 | 50 | 500 | 1000 | 7.4 | 12.5 | 5.2 | 0.1 | 4.6 | 5.0 | 2.9 | 1.1 |
0.9991 | 7.3 | 14.8 | 14.6 | 5.6 | 7.1 | 7.5 | 4.1 | 3.6 | ||||||||
GLE | 0.015 | 0.05 | 200 | 0.9994 | 0.05 | 0.1 | 50 | 100 | 9.0 | 10.5 | −11.7 | 5.4 | 3.4 | 4.6 | 5.1 | 1.4 |
0.9994 | −4.4 | 13.4 | 9.0 | 10.8 | 9.3 | 10.5 | 7.7 | 3.9 | ||||||||
LED | 0.015 | 0.05 | 200 | 0.9994 | 0.05 | 0.1 | 50 | 100 | 8.0 | 10.8 | 7.0 | 2.2 | 8.2 | 4.0 | 3.0 | 0.8 |
0.9995 | −4.6 | −9.2 | 7.3 | 5.6 | 4.2 | 5.3 | 5.3 | 1.4 | ||||||||
LOP | 0.003 | 0.01 | 20 | 0.9996 | 0.01 | 0.02 | 5 | 10 | 7.0 | −2.5 | 7.1 | 9.8 | 4.2 | 6.3 | 2.3 | 2.4 |
0.9998 | 3.0 | 7.0 | −1.4 | 0.5 | 5.5 | 2.0 | 4.6 | 1.7 | ||||||||
MAR | 0.0015 | 0.005 | 20 | 0.9975 | 0.005 | 0.01 | 5 | 10 | −1.7 | 5.0 | 8.1 | 7.4 | 9.3 | 4.8 | 1.7 | 0.6 |
0.9982 | 8.0 | 2.0 | 6.7 | 1.4 | 4.1 | 4.4 | 1.1 | 1.9 | ||||||||
RIL | 0.006 | 0.02 | 20 | 0.9974 | 0.02 | 0.05 | 50 | 100 | 15.0 | 0.0 | 4.0 | 9.4 | 11.9 | 7.1 | 2.1 | 1.4 |
0.9968 | 10.0 | 4.0 | 2.6 | −2.0 | 20.3 | 12.9 | 1.3 | 2.9 | ||||||||
RIT | 0.0006 | 0.002 | 20 | 0.9949 | 0.002 | 0.005 | 5 | 10 | 5.0 | 2.0 | 7.4 | 11.0 | 10.6 | 4.4 | 2.7 | 2.3 |
0.9980 | 0.1 | −2.0 | 0.9 | 1.3 | 0.1 | 4.6 | 4.2 | 1.4 | ||||||||
SAQ | 0.0015 | 0.005 | 20 | 0.9994 | 0.005 | 0.01 | 5 | 10 | −2.0 | 10.0 | −8.0 | −0.8 | 4.6 | 10.2 | 5.8 | 0.9 |
0.9995 | −8.0 | −3.0 | 4.1 | 11.1 | 14.2 | 12.9 | 3.4 | 3.7 | ||||||||
SOF | 0.0015 | 0.005 | 20 | 0.9981 | 0.005 | 0.01 | 5 | 10 | 6.0 | 5.0 | 6.3 | 4.6 | 5.2 | 4.8 | 0.4 | 1.7 |
0.9966 | 6.0 | −2.0 | 2.8 | 1.8 | 8.4 | 2.8 | 1.0 | 1.0 | ||||||||
TNA | 0.0015 | 0.005 | 20 | 0.9997 | 0.005 | 0.01 | 5 | 10 | 8.0 | 9.0 | 2.7 | 0.6 | 4.1 | 2.1 | 1.3 | 1.4 |
0.9994 | 4.0 | −3.0 | 1.1 | 2.8 | 5.3 | 2.8 | 2.2 | 1.1 | ||||||||
TDF | 0.0015 | 0.005 | 20 | 0.9998 | 0.005 | 0.01 | 5 | 10 | 2.0 | −3.0 | 3.7 | 0.3 | 4.4 | 2.8 | 1.9 | 1.7 |
0.9997 | −6.0 | −3.0 | −2.6 | −1.3 | 5.8 | 4.6 | 0.9 | 3.0 | ||||||||
VEL | 0.15 | 0.5 | 500 | 0.9976 | 0.5 | 1 | 100 | 500 | 5.4 | 6.1 | 14.3 | 7.2 | 2.2 | 2.7 | 1.7 | 2.3 |
0.9967 | −2.2 | −8.5 | 14.2 | 4.5 | 7.5 | 4.0 | 1.6 | 3.5 | ||||||||
ZID | 0.006 | 0.02 | 100 | 0.9994 | 0.02 | 0.05 | 50 | 100 | 13.7 | 2.7 | 0.9 | −11.9 | 13.2 | 4.1 | 7.3 | 2.5 |
0.9995 | 13.0 | 4.3 | −8.5 | −5.6 | 15.3 | 7.1 | 4.9 | 7.5 |
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Fical, L.; Khalikova, M.; Kočová Vlčková, H.; Lhotská, I.; Hadysová, Z.; Vokřál, I.; Červený, L.; Švec, F.; Nováková, L. Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes. Molecules 2021, 26, 2123. https://doi.org/10.3390/molecules26082123
Fical L, Khalikova M, Kočová Vlčková H, Lhotská I, Hadysová Z, Vokřál I, Červený L, Švec F, Nováková L. Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes. Molecules. 2021; 26(8):2123. https://doi.org/10.3390/molecules26082123
Chicago/Turabian StyleFical, Luboš, Maria Khalikova, Hana Kočová Vlčková, Ivona Lhotská, Zuzana Hadysová, Ivan Vokřál, Lukáš Červený, František Švec, and Lucie Nováková. 2021. "Determination of Antiviral Drugs and Their Metabolites Using Micro-Solid Phase Extraction and UHPLC-MS/MS in Reversed-Phase and Hydrophilic Interaction Chromatography Modes" Molecules 26, no. 8: 2123. https://doi.org/10.3390/molecules26082123